AAV vectors with high transduction efficiency and uses thereof for gene therapy

ABSTRACT

The present invention provides AAV capsid proteins comprising modification of one or a combination of the surface-exposed lysine, serine, threonine and/or tyrosine residues in the VP3 region. Also provided are rAAV virions comprising the AAV capsid proteins of the present invention, as well as nucleic acid molecules and rAAV vectors encoding the AAV capsid proteins of the present invention. Advantageously, the rAAV vectors and virions of the present invention have improved efficiency in transduction of a variety of cells, tissues and organs of interest, when compared to wild-type rAAV vectors and virions.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 61/647,318, filed May 15, 2012, which isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under a grant awardedfrom the National Institutes of Health under grant number HL097088. Thegovernment has certain rights in the invention.

BACKGROUND OF INVENTION

Major advances in the field of gene therapy have been achieved by usingviruses to deliver therapeutic genetic material. The adeno-associatedvirus (AAV) has attracted considerable attention as a highly effectiveviral vector for gene therapy due to its low immunogenicity and abilityto effectively transduce non-dividing cells. AAV has been shown toinfect a variety of cell and tissue types, and significant progress hasbeen made over the last decade to adapt this viral system for use inhuman gene therapy.

In its normal “wild type” form, recombinant AAV (rAAV) DNA is packagedinto the viral capsid as a single stranded molecule about 4600nucleotides (nt) in length. Following infection of the cell by thevirus, the molecular machinery of the cell converts the single DNAstrand into a double-stranded form.

AAV has many properties that favor its use as a gene deliveryvehicle: 1) the wild type virus is not associated with any pathologichuman condition; 2) the recombinant form does not contain native viralcoding sequences; and 3) persistent transgenic expression has beenobserved in many applications. One of the main obstacles of the genetherapy, the induction of immuno-competition in cellular immuneresponses against vector-derived and transgene-derived epitopes, can beovercome by replication-deficiency and lack of viral proteins expressedby recombinant AAV.

The transduction efficiency of recombinant adeno-associated virusvectors varies greatly in different cells and tissues in vitro and invivo. Systematic studies have been performed to elucidate thefundamental steps in the life cycle of AAV. For example, it has beendocumented that a cellular protein, FKBP52, phosphorylated at tyrosineresidues by epidermal growth factor receptor protein tyrosine kinase(EGFR-PTK), inhibits AAV second-strand DNA synthesis and consequently,transgene expression in vitro as well as in vivo. It has also beendemonstrated that EGFR-PTK signaling modulates the ubiquitin/proteasomepathway-mediated intracellular trafficking as well as FKBP52-mediatedsecond-strand DNA synthesis of AAV vectors. In those studies, inhibitionof EGFR-PTK signaling led to decreased ubiquitination of AAV capsidproteins, which in turn, facilitated nuclear transport by limitingproteasome-mediated degradation of AAV vectors, implicatingEGFR-PTK-mediated phosphorylation of tyrosine residues on AAV capsids.

BRIEF SUMMARY

The present invention provides AAV capsid proteins comprisingmodification of one or a combination of the surface-exposed lysine,serine, threonine and/or tyrosine residues in the VP3 region. Alsoprovided are rAAV virions comprising the AAV capsid proteins of thepresent invention, as well as nucleic acid molecules and rAAV vectorsencoding the AAV capsid proteins of the present invention.Advantageously, the rAAV vectors and virions of the present inventionhave improved efficiency in transduction of a variety of cells, tissuesand organs of interest, when compared to wild-type rAAV vectors andvirions.

In one embodiment, the present invention provides a nucleic acidmolecule comprising a nucleotide sequence encoding an AAV capsidprotein, wherein the VP3 region of the AAV capsid protein comprises anon-lysine residue at a position that corresponds to a lysine residue inthe VP3 region of the capsid protein of the wild-type AAV (e.g., SEQ IDNOs:1-10; in one embodiment, the capsid protein of wild-type AAV2 (SEQID NO:2)), wherein said lysine residue in the VP3 region of thewild-type AAV is selected from the group consisting of K258, K321, K459,K490, K507, K527, K572, K532, K544, K549, K556, K649, K655, K665, andK706.

In one embodiment, the surface-exposed lysine residue corresponding K532of the wild-type AAV2 capsid sequence is modified. In one embodiment,the surface-exposed lysine residue of the AAV capsid is modified intoglutamic acid (E) or arginine (R). In specific embodiments, thesurface-exposed lysine residue corresponding K532 of the wild-type AAV2capsid sequence is modified into arginine (K532R).

In certain embodiments, one or more surface-exposed lysine residuescorresponding to K490, K544, K549, and K556 of the wild-type AAV2 capsidsequence are modified. In certain specific embodiments, one or moresurface-exposed lysine residue corresponding K490, K544, K549, and K556of the wild-type AAV2 capsid sequence are modified into glutamic acid(E).

In one embodiment, the present invention provides AAV2 vectors whereinsurface-exposed lysine residues corresponding to K544 and K556 residuesof the wild-type AAV2 capsid are modified into glutamic acid (E).

In certain embodiments, one or more surface-exposed lysine residuescorresponding to K530, K547, and K569 of the wild-type AAV8 capsidsequence are modified. In certain specific embodiments, one or moresurface-exposed lysine residue corresponding K530, K547, and K569 of thewild-type AAV2 capsid sequence are modified into glutamic acid (E).

In one embodiment, a combination of surface-exposed lysine, serine,threonine and/or tyrosine residues of the AAV capsid is modified,wherein the modification occurs at positions corresponding to(Y444F+Y500F+Y730F+T491V), (Y444F+Y500F+Y730F+T491V+T550V),(Y444F+Y500F+Y730F+T491V+T659V), (T491V+T550V+T659V),(Y440F+Y500F+Y730F), (Y444F+Y500F+Y730F+T491V+S662V), and/or(Y444F+Y500F+Y730F+T491V+T550V+T659V) of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV2 (SEQ ID NO:2)).

In addition, the present invention provides a method for transduction ofcells, tissues, and/or organs of interest, comprising introducing into acell, a composition comprising an effective amount of a rAAV vectorand/or virion of present invention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 1 (AAV1).

SEQ ID NO:2 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 2 (AAV2).

SEQ ID NO:3 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 3 (AAV3).

SEQ ID NO:4 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 4 (AAV4).

SEQ ID NO:5 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 5 (AAV5).

SEQ ID NO:6 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 6 (AAV6).

SEQ ID NO:7 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 7 (AAV7).

SEQ ID NO:8 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 8 (AAV8).

SEQ ID NO:9 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 9 (AAV9).

SEQ ID NO:10 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 10 (AAV10).

SEQ ID NO:11 is a primer sequence useful according to the presentinvention.

SEQ ID NO:12 is a primer sequence useful according to the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a model for AAV2 trafficking inside host cells.

FIG. 2 shows amino acid alignment of the wild-type AAV1-10 capsids. (A)shows amino acid alignment of the wild-type AAV1-10 serotype capsids(SEQ ID NOs:1-10). (B) shows conserved, surface-exposed lysine residuesin the wild-type AAV1-12 capsids, as well as embodiments of amino acidmodifications. The lysine residues conserved among AAV1-12 are shown inbold. (C) shows amino acid alignment of the wild-type AAV1-10 serotypecapsids, as well as surface-exposed tyrosine residues that are conservedamong AAV1-10 capsids (conserved, surface-exposed residues are shown inbold). (D) shows amino acid alignment of the wild-type AAV1-10 serotypecapsids, as well as surface-exposed serine and threonine residues thatare conserved in among AAV1-10 capsids (conserved, surface-exposedresidues are shown in bold).

FIG. 3 shows the effect of various kinase inhibitors on ssAAV and scAAVmediated EGFP expression in HEK293 cells. Cells were pretreated withinhibitors for 1 hr before infection and then transduced with 1×10³vgs/cell. (A) Transgene expression was detected by fluorescencemicroscopy 48 h post infection. (B) Images from three visual fields wereanalyzed as described in Materials and Methods. *P<0.005, **P<0.001 vs.WT AAV2.

FIG. 4 shows an analysis of EGFP expression after transduction of 293cells with individual site-directed AAV2 capsid mutants. Each of the 15surface-exposed serines (S) in AAV2 capsid was substituted with valine(V) and evaluated for its efficiency to mediate trangene expression. (A)EGFP expression analysis at 48 h post-infection at an MOI of 1×10³vgs/cell. (B) Quantitation of transduction efficiency of each of theserine-mutant AAV2 vectors. *P<0.005, **P<0.001 vs. WT AAV2.

FIG. 5 shows an evaluation of the effect of serine substitution atposition 662 in the AAV2 capsid with different amino acids in mediatingtransgene expression. The following 8 serine mutants were generated withdifferent amino acids: S662→Valine (V), S662→Alanine (A),S662→Asparagine (N), S662→Aspartic acid (D), S662→Histidine (H),S662→Isoleucine (I), S662→Leucine (L), and S662→Phenylalanine (F), andtheir transduction efficiency in 293 cells was analyzed. (A) EGFPexpression analysis at 48 h after infection of 293 cells at an MOI of1×10³ vgs/cell. (B) Quantitation of the transduction efficiency of eachof the serine-mutant AAV2 vectors. *P<0.005, **P<0.001 vs. WT AAV2.

FIG. 6 shows an analysis of correlation of transduction efficiency ofAAV2-S662V vectors with p38 MAPK activity in various cell types. (A)Quantitation of the transduction efficiency of WT- and S662V-AAV2vectors in 293, HeLa, NIH3T3, H2.35 and moDCs. (B) Western blot analysisof lysates from different cell lines for p-p38 MAPK expression levels.Total p38 MAPK and GAPDH levels were measured and used as loadingcontrols. *P<0.005, **P<0.001 vs. WT AAV2.

FIG. 7 shows AAV vector-mediated transgene expression inmonocytes-derived dendritic cells (moDCs). (A) Effect of JNK and p38MAPK inhibitors, and site-directed substitution of the serine residue atposition 662 on EGFP expression. (B) Quantitation of the data in (A) at48 h after infection and initiation of maturation. (C) Analysis ofexpression of co-stimulatory markers such as CD80, CD83, CD86 in moDCsinfected with AAV2-S662V vectors at an MOI 5×10⁴ vgs/cell. iDCs—immaturedendritic cells, and mDCs—mature dendritic cells, stimulated withcytokines, generated as described in Materials and Methods, were used asnegative and positive controls, respectively. A representative exampleis shown. *P<0.005, **P<0.001 vs. WT AAV2.

FIG. 8 shows an analysis of hTERT-specific cytotoxic T-lymphocytes(CTLs) killing activity on K562 cells. CTLs were generated aftertransduction of moDCs by AAV2-S662V vectors encoding the truncated humantelomerase (hTERT). AAV2-S662V-EGFP vector-traduced moDCs were used togenerate non-specific CTLs. Pre-stained with3,3-dioctadecyloxacarbocyanine (DiOC18(3)), a green fluorescent membranestain, 1×10⁵ target K562 cells were co-cultured overnight with differentratios of CTLs (80:1, 50:1, 20:1, 10:1, 5:1). Membrane-permeable nucleicacid counter-stain, propidium iodide, was added to label the cells withcompromised plasma membranes. Percentages of killed, doublestain-positive cells were analyzed by flow cytometry.

FIG. 9 shows packaging and transduction efficiencies of variousserine-valine mutant AAV2 vectors relative to wild-type (WT) AAV2vectors. Briefly, vector packaging and infectivity assays were performedat least twice for each of the mutant-AAV vectors. The packagingefficiency was determined by quantitative PCR analyses. The transductionefficiency was estimated by fluorescence intensity. * No fluorescencewas detected at the MOI tested.

FIG. 10 shows packaging and transduction efficiencies of serine-mutantvectors replaced with various amino acids relative to wild-type (WT)AAV2 vectors. The packaging and infectivity assays were performed asdescribed in FIG. 9. V=Valine; A=Alanine; D=Aspartic acid;F=Phenylalanine H=Histidine; N=Asparagine; L=Leucine; and I=Isoleucine.

FIG. 11 shows that site-directed mutagenesis of surface-exposed serineresidues increase transduction efficiency of 293 cells by scAAV vectors.

FIG. 12 shows that site-directed mutagenesis of surface-exposedthreonine residues increase transduction efficiency of 293 cells byscAAV vectors.

FIG. 13 shows that site-directed mutagenesis of a combination ofsurface-exposed serine, threonine and/or tyrosine residues increasetransduction efficiency of H2.35 cells by scAAV vectors.

FIG. 14 shows that site-directed mutagenesis of a combination ofsurface-exposed serine, threonine and/or tyrosine residues increasetransduction efficiency of monocyte-derived dendritic cells by scAAVvectors.

FIG. 15 shows transduction efficiency of AAV2 lysine mutants (MOI 2000)in HeLa and HEK293 cells in vitro. The relative fold-increase in geneexpression is shown as inserts.

FIG. 16(A) shows AAV vector-induced innate immune response in mice invivo. Gene expression profiling of innate immune mediators wasperformed, and data for fold changes in gene expression at the 2 hrstime-point comparing AAV vectors with Bay11 (hatched or open bars) withAAV vectors without Bay11 (black or grey bars) are shown. The minimalthreshold fold-increase (horizontal black line) was 2.5. (B) Westernblot analysis of liver homogenates from mice 9 hrs followingmock-injections, or injections with scAAV vectors, with and withoutprior administration of Bay11. Samples were analyzed by using anti-p52antibody for detection of NF-κB signaling in response to AAV exposure.Anti-β-actin antibody was used as a loading control. (C) Humoralresponse to AAV vectors in the absence or presence of NF-κB inhibitor.Anti-AAV2 IgG2a levels were determined in peripheral blood from mice atday 10 following injections with scAAV vectors, with and without prioradministration of Bay11 (n=4 each). (D) Transgene expression in murinehepatocytes 10 days post-injection of 1×10¹¹ vgs each of WT-scAAV-EGFPor TM-scAAV-EFGP vectors/animal via the tail-vein. (E) Quantitativeanalyses of data from (D).

FIG. 17 shows AAV3-mediated transgene expression in T47D and T47D+hHGFRcells. (A) Equivalent numbers of T47D and T47D+hHGFR cells were infectedwith various indicated multiplicity-of-infection of scAAV3-CBAp-EGFPvectors under identical conditions. Transgene expression was determinedby fluorescence microscopy 72 hrs post-infection. (B) T47D+hHGFR cellswere transduced with 2,000 vgs/cell of scAAV3 vectors in the absence orthe presence of 5 μg/ml of hHGF. Transgene expression was determined asabove. (C) The effect of HGFR kinase-specific inhibitor, BMS-777607(BMS), on AAV3-mediated transgene expression is shown. T47D andT47D+hHGFR cells were mock-treated or pretreated with BMS for 2 hrs.Whole-cell lysates were prepared and analyzed on Western blots usingvarious indicated primary antibodies. β-actin was used as a loadingcontrol. (D) Transduction efficiency of WT and single, double, andtriple tyrosine-mutant AAV3 vectors. Huh7 cells were transduced with WTor various indicated Y-F mutant scAAV3-CBAp-EGFP vectors under identicalconditions.

FIG. 18 shows the transduction efficiency of WT- and lysine-mutantscAAV2 vectors in HeLa cells in vitro (2,000 vgs/cell; 48 hrs).

FIG. 19 shows the transduction efficiency of WT- and lysine-mutantscAAV2 vectors in primary hepatocytes in vivo (C57BL/6 mice; 1×10¹⁰scAAV-2-CBAp-EGFP vectors; tail-vein injections; 2-weeks).

FIG. 20 shows the transduction efficiency of WT-, lysine-, andtyrosine-mutant scAAV2 vectors in primary hepatocytes in vivo (C57BL/6mice; 1×10¹⁰ scAAV-2-CBAp-Fluc vectors; tail-vein injections; 2-weeks).

FIG. 21 shows the quantification of transgene expression by AAV2-K544E,AAV2-K556E, AAV2-K544/566E, and AAV2-Y444/500/730F.

FIG. 22 shows the transduction efficiency of WT- and lysine-mutantscAAV8 vectors in murine hepatocytes in vivo (C57BL/6 mice; 1×10¹⁰scAAV-2-CBAp-Fluc vectors; tail-vein injections; 2-weeks) (experiment1).

FIG. 23 shows the quantification of transgene expression by AAV8-K530E,AAV8-K547E, and AAV8-K569E.

FIG. 24 shows the transduction efficiency of WT- and lysine-mutantscAAV8 vectors in primary hepatocytes in vivo (C57BL/6 mice; 1×10¹⁰scAAV-2-CBAp-Fluc vectors; tail-vein injections; 2-weeks) (experiment 2)

FIG. 25 shows the quantification of transgene expression by AAV8-K530E,AAV8-K547E, and AAV8-K569E.

DETAILED DISCLOSURE

The present invention provides AAV capsid proteins comprisingmodification of one or a combination of the surface-exposed lysine,serine, threonine and/or tyrosine residues in the VP3 region. Alsoprovided are rAAV virions comprising the AAV capsid proteins of thepresent invention, as well as nucleic acid molecules and rAAV vectorsencoding the AAV capsid proteins of the present invention.Advantageously, the rAAV vectors and virions of the present inventionhave improved efficiency in transduction of a variety of cells, tissuesand organs of interest and/or reduces host immune responses to thevectors, when compared to wild-type rAAV vectors and virions.

In one embodiment, the present invention provides a nucleic acidmolecule comprising a nucleotide sequence encoding an AAV capsidprotein, wherein the VP3 region of the AAV capsid protein comprises anon-lysine residue at one or more positions that correspond to one ormore lysine residues in the VP3 region of the capsid protein of thewild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsidprotein of wild-type AAV2 (SEQ ID NO:2)), wherein said lysine residue(s)in the VP3 region of the wild-type AAV is selected from the lysineresidues as indicated in FIG. 2B.

In a specific embodiment, one or more surface-exposed lysine residuecorresponding to one or more lysine residues selected from the groupconsisting of K258, K321, K459, K490, K507, K527, K572, K532, K544,K549, K556, K649, K655, K665, and K706 of the wild-type AAV capsidprotein (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)) are modified.

In one embodiment, the surface-exposed lysine residue corresponding toK532 of the wild-type AAV2 capsid sequence is modified. In oneembodiment, the surface-exposed lysine residue of the AAV capsid ismodified into glutamic acid (E) or arginine (R). In one specificembodiment, the surface-exposed lysine residue corresponding to K532 ofthe wild-type AAV2 capsid sequence is modified into arginine (K532R).

In certain embodiments, one or more surface-exposed lysine residuescorresponding to K490, K544, K549, and K556 of the wild-type AAV2 capsidsequence are modified. In certain specific embodiments, one or moresurface-exposed lysine residue corresponding K490, K544, K549, and K556of the wild-type AAV2 capsid sequence are modified into glutamic acid(E).

In one embodiment, the present invention provides AAV2 vectors whereinsurface-exposed lysine residues corresponding to K544 and K556 residuesof the wild-type AAV2 capsid are modified into glutamic acid (E).

In certain embodiments, one or more surface-exposed lysine residuescorresponding to K530, K547, and K569 of the wild-type AAV8 capsidsequence are modified. In certain specific embodiments, one or moresurface-exposed lysine residue corresponding K530, K547, and K569 of thewild-type AAV2 capsid sequence are modified into glutamic acid (E) orarginine (R).

In addition, the present invention provides a method for transduction ofcells, tissues, and/or organs of interest, comprising introducing into acell, a composition comprising an effective amount of a rAAV vectorand/or virion of the present invention.

Phosphorylation of surface-exposed lysine, serine, threonine and/ortyrosine residues on the AAV capsids can result in theubiquitination/proteasomal degradation of the vectors. Serine/threonineprotein kinases are involved in a wide variety of cellular processesincluding cell differentiation, transcription regulation, anddevelopment. Phosphorylation of the surface-exposed serine and/orthreonine residues on the viral capsid induces proteasome-mediateddegradation of the vectors and reduces vector transduction efficiency.Cellular epidermal growth factor receptor protein tyrosine kinase(EGFR-PTK) also phosphorylates capsids at surface tyrosine residues,and, thus negatively impacts nuclear transport and subsequent transgeneexpression by recombinant AAV2 vectors.

Surface-exposed lysine, serine, threonine and/or tyrosine residues onthe AAV capsids are identified (FIG. 2). For instance, the VP3 region ofthe capsid protein of the wild-type AAV2 contains varioussurface-exposed lysine (K) residues (K258, K321, K459, K490, K507, K527,K572, K532, K544, K549, K556, K649, K655, K665, K706), surface-exposedserine (S) residues (S261, S264, S267, S276, S384, S458, S468, S492,S498, S578, S658, S662, S668, S707, S721), surface-exposed threonine (T)residues (T251, T329, T330, T454, T455, T503, T550, T592, T581, T597,T491, T671, T659, T660, T701, T713, T716), and surface-exposed tyrosineresidues (Y252, Y272, Y444, Y500, Y700, Y704, Y730). As shown in FIG. 2,these surface-exposed lysine, serine, threonine and/or tyrosine residuesof the capsids are highly conserved among AAV1-12.

Site-directed mutagenesis of the surface-exposed lysine, serine,threonine and/or tyrosine residues was performed and the results showthat modification or substitution of one or a combination of thesurface-exposed residues can enable the AAV vector to bypass theubiquitination and proteasome-mediated degradation steps, therebyyielding novel AAV vectors with high-efficiency transduction.Substitution of surface exposed tyrosine residues on AAV capsids permitsthe vectors to escape ubiquitination, and thus, inhibitsproteasome-mediated degradation. Although phosphorylated AAV vectorscould enter cells as efficiently as their unphosphorylated counterparts,their transduction efficiency was significantly reduced. This reductionwas not due to impaired viral second-strand DNA synthesis sincetransduction efficiency of both single-stranded AAV (ssAAV) andself-complementary AAV (rAAV) vectors was decreased.

Recombinant AAV vectors containing point mutations in surface exposedtyrosine residues confer higher transduction efficiency at lower doses,when compared to the wild-type (WT) AAV vectors.

In addition, in accordance of the present invention, (i) site-directedmutagenesis of the 15 surface-exposed serine (S) residues on the AAV2capsid with valine (V) residues leads to improved transductionefficiency of S458V, S492V, and S662V mutant vectors compared with theWT AAV2 vector; (ii) the S662V mutant vector efficiently transducesprimary human monocyte-derived dendritic cells (moDCs), a cell type notreadily amenable to transduction by the conventional AAV vectors; (iii)high-efficiency transduction of moDCs by S662V mutant does not induceany phenotypic changes in these cells; and (iv) recombinant S662V-rAAVvectors carrying a truncated human telomerase (hTERT) gene transducedDCs result in rapid, specific T-cell clone proliferation and generationof robust CTLs, which lead to specific cell lysis of K562 cells. Theresults demonstrate that the serine-modified rAAV2 vectors of thepresent invention result in high-efficiency transduction of moDCs.

In the setting of tumor immunotherapy, the time of T-cell activation andthe potency and longevity of CD8 T cell responses are crucial factors indetermining the therapeutic outcome. In accordance with the presentinvention, increased transduction efficiency of moDC by theserine-mutant AAV2 vectors results in superior priming of T-cells. Humantelomerase was used as a specific target since clinical studies haveshown that human telomerase is an attractive candidate for a broadlyexpressed rejection antigen for many cancer patients. In addition,transduction efficiency of the S662V mutant vector was further augmentedby pre-treatment of cells with specific inhibitors of JNK and p38 MAPK,indicating that one or more surface-exposed threonine (T) residues onAAV2 capsids are most likely phosphorylated by these kinases.

Recombinant AAV Vectors and Virions

One aspect of the invention provides AAV capsid proteins comprisingmodification of one or a combination of the surface-exposed lysine,serine, threonine and/or tyrosine residues in the VP3 region. Alsoprovided are rAAV virions comprising the AAV capsid proteins of thepresent invention, as well as nucleic acid molecules and rAAV vectorsencoding the AAV capsid proteins of the present invention.Advantageously, the rAAV vectors and virions of the present inventionhave improved efficiency in transduction of a variety of cells, tissuesand organs of interest, when compared to wild-type rAAV vectors andvirions.

In one embodiment, the present invention provides a nucleic acidmolecule comprising a nucleotide sequence encoding an AAV capsidprotein, wherein the AAV capsid protein comprises modification of one ora combination of the surface-exposed lysine, serine, threonine and/ortyrosine residues in the VP3 region. Advantageously, modification of thesurface-exposed lysine, serine, threonine and/or tyrosine residuesprevents or reduces the level of ubiquitination of the AAV vectors, and,thereby, prevents or reduces the level of proteasome-mediateddegradation. In addition, modification of the surface-exposed lysine,serine, threonine and/or tyrosine residues in accordance with thepresent invention improves transduction efficiency.

In one embodiment, the nucleic acid molecule comprising a nucleotidesequence encoding an AAV capsid protein, wherein the VP3 region of theAAV capsid protein comprises one or a combination of the followingcharacteristics:

(a)

(i) a non-lysine residue at one or more positions that correspond to alysine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)), wherein said lysine residue in the VP3region of the wild-type AAV is selected from the group consisting ofK258, K321, K459, K490, K507, K527, K572, K532, K544, K549, K556, K649,K655, K665, and K706, wherein said non-lysine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(ii) a chemically-modified lysine residue at one or more positions thatcorrespond to a lysine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein said lysineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of K258, K321, K459, K490, K507, K527, K572, K532,K544, K549, K556, K665, K649, K655, and K706, wherein saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(iii) a non-lysine residue at one or more positions that correspond to alysine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV8 (SEQ ID NO:8)), wherein said lysine residue in the VP3region of the wild-type AAV is selected from the group consisting ofK530, K547, and K569, wherein said non-lysine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(iv) a chemically-modified lysine residue at one or more positions thatcorrespond to a lysine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV8 (SEQ ID NO:8)), wherein said lysineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of K530, K547, and K569, wherein saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(b)

(i) a non-serine residue at one or more positions that correspond to aserine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)), wherein said serine residue in the VP3region of the wild-type AAV is selected from the group consisting ofS261, S264, S267, S276, S384, S458, S468, S492, S498, S578, S658, S662,S668, S707, and S721, wherein said non-serine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(ii) a chemically-modified serine residue at one or more positions thatcorrespond to a serine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein said serineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of S261, S264, S267, S276, S384, S458, S468, S492,S498, S578, S658, S662, S668, S707, and S721, wherein saidchemically-modified serine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(c)

(i) a non-threonine residue at one or more positions that correspond toa threonine residue in the VP3 region of the capsid protein of thewild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsidprotein of wild-type AAV2 (SEQ ID NO:2)), wherein said threonine residuein the VP3 region of the wild-type AAV is selected from the groupconsisting of T251, T329, T330, T454, T455, T503, T550, T592, T581,T597, T491, T671, T659, T660, T701, T713, and T716, wherein saidnon-threonine residue does not result in phosphorylation and/orubiquitination of an AAV vector; and/or

(ii) a chemically-modified threonine residue at one or more positionsthat correspond to a threonine residue in the VP3 region of the capsidprotein of the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment,the capsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein saidthreonine residue in the VP3 region of the wild-type AAV is selectedfrom the group consisting of T251, T329, T330, T454, T455, T503, T550,T592, T581, T597, T491, T671, T659, T660, T701, T713, and T716, whereinsaid chemically-modified threonine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(d)

(i) a non-tyrosine residue at one or more positions that correspond to atyrosine residue in the VP3 region of the capsid protein of thewild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsidprotein of wild-type AAV2 (SEQ ID NO:2)), wherein said tyrosine residuein the VP3 region of the wild-type AAV is selected from the groupconsisting of Y252, Y272, Y444, Y500, Y700, Y704, and Y730, wherein saidnon-tyrosine residue does not result in phosphorylation and/orubiquitination of an AAV vector; and/or

(ii) a chemically-modified tyrosine residue at one or more positionsthat correspond to a tyrosine residue in the VP3 region of the capsidprotein of the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment,the capsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein saidtyrosine residue in the VP3 region of the wild-type AAV is selected fromthe group consisting of Y252, Y272, Y444, Y500, Y700, Y704, and Y730,wherein said chemically-modified tyrosine residue does not result inphosphorylation and/or ubiquitination of an AAV vector.

In another embodiment, the present invention provides a nucleic acidmolecule comprising a nucleotide sequence encoding an AAV capsidprotein, wherein one or more of surface-exposed lysine, serine,threonine and/or tyrosine residues in the VP3 region are modified asfollows:

(a)

(i) at least one lysine residue in the VP3 region is chemically modifiedor is modified into a non-lysine residue, wherein the modified residuecorresponds to K258, K321, K459, K490, K507, K527, K572, K532, K544,K549, K556, K649, K655, or K706 of the wild-type AAV capsid sequence(e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)), wherein said non-lysine residue or saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector; and/or

(ii)

at least one lysine residue in the VP3 region is chemically modified oris modified into a non-lysine residue, wherein the modified residuecorresponds to K530, K547, or K569 of the wild-type AAV capsid sequence(e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV8 (SEQ ID NO:8)), wherein said non-lysine residue or saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(b) at least one serine residue in the VP3 region is chemically modifiedor is modified into a non-serine residue, wherein the modified residuecorresponds to S261, S264, S267, S276, S384, S458, S468, S492, S498,S578, S658, S662, S668, S707, or S721 of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV2 (SEQ ID NO:2)), wherein said non-serine residue orsaid chemically-modified serine residue does not result inphosphorylation and/or ubiquitination of an AAV vector;

(c) at least one threonine residue in the VP3 region is chemicallymodified or is modified into a non-threonine residue, wherein themodified residue corresponds to T251, T329, T330, T454, T455, T503,T550, T592, T581, T597, T491, T671, T659, T660, T701, T713, or T716 ofthe wild-type AAV capsid sequence (e.g., SEQ ID NOs:1-10; in oneembodiment, the capsid protein of wild-type AAV2 (SEQ ID NO:2)), whereinsaid non-threonine residue or said chemically-modified threonine residuedoes not result in phosphorylation and/or ubiquitination of an AAVvector; and

(d) at least one tyrosine residue in the VP3 region is chemicallymodified or is modified into a non-tyrosine residue, wherein themodified residue corresponds to Y252, Y272, Y444, Y500, Y700, Y704, orY730 the wild-type AAV capsid sequence (e.g., SEQ ID NOs:1-10; in oneembodiment, the capsid protein of wild-type AAV2 (SEQ ID NO:2)), whereinsaid non-tyrosine residue or said chemically-modified tyrosine residuedoes not result in phosphorylation and/or ubiquitination of an AAVvector.

The present invention also provides AAV VP3 capsid proteins havingmodification of one or more surface-exposed lysine, serine, threonineand/or tyrosine residues. In one embodiment, the present inventionprovides an AAV VP3 capsid protein comprising one or a combination ofthe following characteristics:

(a)

(i) a non-lysine residue at one or more positions that correspond to alysine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)), wherein said lysine residue in the VP3region of the wild-type AAV is selected from the group consisting ofK258, K321, K459, K490, K507, K527, K572, K532, K544, K549, K556, K649,K655, K665, and K706, wherein said non-lysine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(ii) a chemically-modified lysine residue at one or more positions thatcorrespond to a lysine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein said lysineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of K258, K321, K459, K490, K507, K527, K572, K532,K544, K549, K556, K649, K655, K665, and K706, wherein saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(iii) a non-lysine residue at one or more positions that correspond to alysine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV8 (SEQ ID NO:8)), wherein said lysine residue in the VP3region of the wild-type AAV is selected from the group consisting ofK530, K547, and K569, wherein said non-lysine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(iv) a chemically-modified lysine residue at one or more positions thatcorrespond to a lysine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV8 (SEQ ID NO:8)), wherein said lysineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of K530, K547, and K569, wherein saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(b)

(i) a non-serine residue at one or more positions that correspond to aserine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)), wherein said serine residue in the VP3region of the wild-type AAV is selected from the group consisting ofS261, S264, S267, S276, S384, S458, S468, S492, S498, S578, S658, S662,S668, S707, and S721, wherein said non-serine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(ii) a chemically-modified serine residue at one or more positions thatcorrespond to a serine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein said serineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of S261, S264, S267, S276, S384, S458, S468, S492,S498, S578, S658, S662, S668, S707, and S721, wherein saidchemically-modified serine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(c)

(i) a non-threonine residue at one or more positions that correspond toa threonine residue in the VP3 region of the capsid protein of thewild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsidprotein of wild-type AAV2 (SEQ ID NO:2)), wherein said threonine residuein the VP3 region of the wild-type AAV is selected from the groupconsisting of T251, T329, T330, T454, T455, T503, T550, T592, T581,T597, T491, T671, T659, T660, T701, T713, and T716, wherein saidnon-threonine residue does not result in phosphorylation and/orubiquitination of an AAV vector; and/or

(ii) a chemically-modified threonine residue at one or more positionsthat correspond to a threonine residue in the VP3 region of the capsidprotein of the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment,the capsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein saidthreonine residue in the VP3 region of the wild-type AAV is selectedfrom the group consisting of T251, T329, T330, T454, T455, T503, T550,T592, T581, T597, T491, T671, T659, T660, T701, T713, and T716, whereinsaid chemically-modified threonine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(d)

(i) a non-tyrosine residue at one or more positions that correspond to atyrosine residue in the VP3 region of the capsid protein of thewild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsidprotein of wild-type AAV2 (SEQ ID NO:2)), wherein said tyrosine residuein the VP3 region of the wild-type AAV is selected from the groupconsisting of Y252, Y272, Y444, Y500, Y700, Y704, and Y730, wherein saidnon-tyrosine residue does not result in phosphorylation and/orubiquitination of an AAV vector; and/or

(ii) a chemically-modified tyrosine residue at one or more positionsthat correspond to a tyrosine residue in the VP3 region of the capsidprotein of the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment,the capsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein saidtyrosine residue in the VP3 region of the wild-type AAV is selected fromthe group consisting of Y252, Y272, Y444, Y500, Y700, Y704, and Y730,wherein said chemically-modified tyrosine residue does not result inphosphorylation and/or ubiquitination of an AAV vector.

In another embodiment, the present invention provides an AAV capsidprotein, wherein one or more of surface-exposed lysine, serine,threonine and/or tyrosine residues in the VP3 region are modified asfollows:

(a)

(i) at least one lysine residue in the VP3 region is chemically modifiedor is modified into a non-lysine residue, wherein the modified residuecorresponds to K258, K321, K459, K490, K507, K527, K572, K532, K544,K549, K556, K649, K655, K665, or K706 of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV2 (SEQ ID NO:2)), wherein said non-lysine residue orsaid chemically-modified lysine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(ii)

at least one lysine residue in the VP3 region is chemically modified oris modified into a non-lysine residue, wherein the modified residuecorrespond to K530, K547, or K569 of the wild-type AAV capsid sequence(e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV8 (SEQ ID NO:8)), wherein said non-lysine residue or saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(b) at least one serine residue in the VP3 region is chemically modifiedor is modified into a non-serine residue, wherein the modified residuecorrespond to S261, S264, S267, S276, S384, S458, S468, S492, S498,S578, S658, S662, S668, S707, or S721 of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV2 (SEQ ID NO:2)), wherein said non-serine residue orsaid chemically-modified serine residue does not result inphosphorylation and/or ubiquitination of an AAV vector;

(c) at least one threonine residue in the VP3 region is chemicallymodified or is modified into a non-threonine residue, wherein themodified residue correspond to T251, T329, T330, T454, T455, T503, T550,T592, T581, T597, T491, T671, T659, T660, T701, T713, or T716 of thewild-type AAV capsid sequence (e.g., SEQ ID NOs:1-10; in one embodiment,the capsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein saidnon-threonine residue or said chemically-modified threonine residue doesnot result in phosphorylation and/or ubiquitination of an AAV vector;and

(d) at least one tyrosine residue in the VP3 region is chemicallymodified or is modified into a non-tyrosine residue, wherein themodified residue correspond to Y252, Y272, Y444, Y500, Y700, Y704, orY730 the wild-type AAV capsid sequence (e.g., SEQ ID NOs:1-10; in oneembodiment, the capsid protein of wild-type AAV2 (SEQ ID NO:2)), whereinsaid non-tyrosine residue or said chemically-modified tyrosine residuedoes not result in phosphorylation and/or ubiquitination of an AAVvector. As shown in FIG. 2, surface-exposed lysine, serine, threonineand/or tyrosine residues located in the VP3 region of the capsid proteinare highly conserved among various AAV serotypes (AAV1 to 12). In oneembodiment, the nucleic acid molecule comprising a nucleotide sequenceencoding an AAV capsid protein, wherein the AAV serotype is selectedfrom AAV1 to 12. In certain embodiments, the wild-type AAV capsidprotein has an amino acid sequence selected from SEQ ID NOs: 1-10.

In one specific embodiment, the nucleic acid molecule comprises anucleotide sequence encoding an AAV2 capsid protein. Theadeno-associated virus 2 (AAV2) is a non-pathogenic human parvovirus.Recombinant AAV2 vectors have been shown to transduce a wide variety ofcells and tissues in vitro and in vivo, and are currently in use inPhase I/II clinical trials for gene therapy of a number of diseases suchas cystic fibrosis, alpha-1 antitrypsin deficiency, Parkinson's disease,Batten's disease, and muscular dystrophy.

In one embodiment, the present invention provides an AAV capsid protein,wherein the AAV capsid protein comprises the amino acid sequence of thecapsid protein of the wild-type AAV2 (SEQ ID NO:2) except that one ormore of the amino acid residues of the wild-type AAV2 capsid aremodified as follows:

(a) at least one lysine residue of the wild-type AAV2 capsid sequenceselected from the group consisting of K258, K321, K459, K490, K507,K527, K572, K532, K544, K549, K556, K649, K655, K665, and K706 ismodified into a non-lysine residue, or said lysine residue is chemicallymodified so that said non-lysine residue or said chemically-modifiedlysine residue does not result in phosphorylation and/or ubiquitinationof an AAV vector;

(b) at least one serine residue of the wild-type AAV2 capsid sequenceselected from the group consisting of S261, S264, S267, S276, S384,S458, S468, S492, S498, S578, S658, S662, S668, S707, and S721 ismodified into a non-serine residue, or said serine residue is chemicallymodified so that said non-serine residue or said chemically-modifiedserine residue does not result in phosphorylation and/or ubiquitinationof an AAV vector;

(c) at least one threonine residue of the wild-type AAV2 capsid sequenceselected from the group consisting of T251, T329, T330, T454, T455,T503, T550, T592, T581, T597, T491, T671, T659, T660, T701, T713, andT716 is modified into a non-threonine residue, or said threonine residueis chemically modified so that said non-threonine residue or saidchemically-modified threonine residue does not result in phosphorylationand/or ubiquitination of an AAV vector; and/or

(d) at least one tyrosine residue of the wild-type AAV2 capsid sequenceselected from the group consisting of Y252, Y272, Y444, Y500, Y700,Y704, and Y730 is modified into a non-threonine residue is modified intoa non-tyrosine residue, or said tyrosine residue is chemically modifiedso that said non-tyrosine residue or said chemically-modified tyrosineresidue does not result in phosphorylation and/or ubiquitination of anAAV vector. In one embodiment, a surface-exposed lysine residuecorresponding to a lysine residue selected from K532, K459, K490, K544,K549, K556, K527, K490, K143, or K137 of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV2 (SEQ ID NO:2)) is modified into a non-lysine residueand/or is chemically modified so that said non-lysine residue or saidmodified lysine residue does not result in phosphorylation and/orubiquitination of an AAV vector.

In another embodiment, the present invention provides an AAV capsidprotein, wherein the AAV capsid protein comprises the amino acidsequence of the capsid protein of the wild-type AAV8 (SEQ ID NO:8)except that one or more surface-exposed lysine residues corresponding toK530, K547, and K569 of the wild-type AAV8 capsid are modified into anon-lysine residue (such as, glutamic acid (E), arginine (R)) and/or aremodified chemically modified, wherein said non-lysine residue or saidmodified lysine residue does not result in phosphorylation and/orubiquitination of an AAV vector. In certain embodiments, thesurface-exposed lysine residues of AAV sequence are modified intoglutamic acid (E), arginine (R), serine (S), or isoleucine (I) to avoidin phosphorylation and/or ubiquitination of the AAV vector.

The present invention also provides a nucleic acid molecule comprises anucleotide sequence encoding an AAV capsid protein (e.g., VP3) of thepresent invention.

In one specific embodiment, the surface-exposed lysine residuecorresponding to K532 of the wild-type AAV2 capsid sequence is modified.In one embodiment, the surface-exposed lysine residue of the AAV capsidis modified into glutamic acid (E) or arginine (R). In one specificembodiment, the surface-exposed lysine residue corresponding to K532 ofthe wild-type AAV2 capsid sequence is modified into arginine (K532R).

In one embodiment, at least one surface-exposed lysine residue of an AAVcapsid corresponding to a lysine position of a wild-type AAV2 capsidsequence is modified as indicated in FIG. 2B.

In one embodiment, at least one surface-exposed serine residuecorresponding to a serine residue selected from S662, S261, S468, S458,S276, S658, S384, or S492 of the wild-type AAV capsid sequence (e.g.,SEQ ID NOs:1-10; in one embodiment, the capsid protein of wild-type AAV2(SEQ ID NO:2)) is modified into a non-serine residue and/or ischemically modified so that said non-serine residue or said modifiedserine residue does not result in phosphorylation and/or ubiquitinationof an AAV vector.

In one specific embodiment, the surface-exposed serine residuecorresponding S662 of the wild-type AAV2 capsid sequence is modified. Inone embodiment, the surface-exposed serine residue of the AAV capsid ismodified into valine (V), aspartic acid (D), or histidine (H). In onespecific embodiment, the surface-exposed serine residue corresponding toS662 of the wild-type AAV2 capsid sequence is modified into valine(S662V).

In one embodiment, a surface-exposed threonine residue corresponding toa threonine residue selected from T455, T491, T550, T659, or T671 of thewild-type AAV capsid sequence (e.g., SEQ ID NOs:1-10; in one embodiment,the capsid protein of wild-type AAV2 (SEQ ID NO:2)) is modified into anon-threonine residue and/or is chemically modified so that saidnon-threonine residue or said modified threonine residue does not resultin phosphorylation and/or ubiquitination of an AAV vector.

In one specific embodiment, the surface-exposed threonine residuecorresponding to T491 of the wild-type AAV2 capsid sequence is modified.In one embodiment, the surface-exposed threonine residue of the AAVcapsid is modified into valine (V). In one specific embodiment, thesurface-exposed threonine residue corresponding to T491 of the wild-typeAAV2 capsid sequence is modified into valine (T491V).

In one embodiment, the AAV vector comprises a modification ofsurface-exposed threonine residues at positions corresponding to(T550V+T659V+T491V) of the wild-type AAV capsid sequence (e.g., SEQ IDNOs:1-10; in one embodiment, the capsid protein of wild-type AAV2 (SEQID NO:2)). In one embodiment, a surface-exposed tyrosine residuecorresponding to a tyrosine residue selected from Y252, Y272, Y444,Y500, Y704, Y720, Y730, or Y673 of the wild-type AAV capsid sequence(e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)) is modified into a non-tyrosine residueand/or is chemically modified so that said non-tyrosine residue or saidmodified tyrosine residue does not result in phosphorylation and/orubiquitination of an AAV vector.

In one embodiment, the surface-exposed tyrosine residue of the AAVcapsid is modified into phenylalanine (F). In one embodiment, the AAVvector comprises a modification of surface-exposed tyrosine residues atpositions corresponding to (Y730F+Y500F+Y444F) of the wild-type AAVcapsid sequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsidprotein of wild-type AAV2 (SEQ ID NO:2)).

In one embodiment, a combination of surface-exposed lysine, serine,threonine and/or tyrosine residues of the AAV capsid is modified,wherein the modification occurs at positions corresponding to(Y444F+Y500F+Y730F+T491V), (Y444F+Y500F+Y730F+T491V+T550V),(Y444F+Y500F+Y730F+T491V+T659V), (T491V+T550V+T659V),(Y440F+Y500F+Y730F), (Y444F+Y500F+Y730F+T491V+S662V), and/or(Y444F+Y500F+Y730F+T491V+T550V+T659V) of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV2 (SEQ ID NO:2)). Also provided are AAV capsid proteinsencoded by the nucleic acid molecules of the present invention.

In one embodiment, the present invention provides a recombinantadeno-associated viral (rAAV) vector comprising a nucleic acid sequencethat encodes an AAV capsid protein of the invention.

In another embodiment, the present invention provides a rAAV virioncomprising an AAV capsid protein of the invention.

In one embodiment, the rAAV vector and virion has enhanced transductionefficiency, when compared to the wild-type rAAV vector and virion. Inanother embodiment, the rAAV vector and virion is capable of efficienttransduction of cells, tissues, and/or organs of interest.

In one embodiment, the rAAV vector further comprises a transgene (alsoreferred to as a heterologous nucleic acid molecule) operably linked toa promoter and optionally, other regulatory elements. In one embodiment,the transgene encodes a therapeutic agent of interest.

Exemplary promoters include one or more heterologous, tissue-specific,constitutive or inducible promoters, including, but not limited to, apromoter selected from the group consisting of cytomegalovirus (CMV)promoters, desmin (DES), beta-actin promoters, insulin promoters,enolase promoters, BDNF promoters, NGF promoters, EGF promoters, growthfactor promoters, axon-specific promoters, dendrite-specific promoters,brain-specific promoters, hippocampal-specific promoters,kidney-specific promoters, elafin promoters, cytokine promoters,interferon promoters, growth factor promoters, alpha-1 antitrypsinpromoters, brain-specific promoters, neural cell-specific promoters,central nervous system cell-specific promoters, peripheral nervoussystem cell-specific promoters, interleukin promoters, serpin promoters,hybrid CMV promoters, hybrid .beta.-actin promoters, EF1 promoters, U1apromoters, U1b promoters, Tet-inducible promoters and VP16-LexApromoters. In exemplary embodiments, the promoter is a mammalian oravian beta-actin promoter.

Exemplary enhancer sequences include, but are not limited to, one ormore selected from the group consisting of CMV enhancers, syntheticenhancers, liver-specific enhancers, vascular-specific enhancers,brain-specific enhancers, neural cell-specific enhancers, lung-specificenhancers, muscle-specific enhancers, kidney-specific enhancers,pancreas-specific enhancers, and islet cell-specific enhancers.

Exemplary therapeutic agents include, but are not limited to, an agentselected from the group consisting of polypeptides, peptides,antibodies, antigen binding fragments, ribozymes, peptide nucleic acids,siRNA, RNAi, antisense oligonucleotides and antisense polynucleotides.

In exemplary embodiments, the rAAV vectors of the invention will encodea therapeutic protein or polypeptide selected from the group consistingof adrenergic agonists, anti-apoptosis factors, apoptosis inhibitors,cytokine receptors, cytokines, cytotoxins, erythropoietic agents,glutamic acid decarboxylases, glycoproteins, growth factors, growthfactor receptors, hormones, hormone receptors, interferons,interleukins, interleukin receptors, kinases, kinase inhibitors, nervegrowth factors, netrins, neuroactive peptides, neuroactive peptidereceptors, neurogenic factors, neurogenic factor receptors, neuropilins,neurotrophic factors, neurotrophins, neurotrophin receptors,N-methyl-D-aspartate antagonists, plexins, proteases, proteaseinhibitors, protein decarboxylases, protein kinases, protein kinsaseinhibitors, proteolytic proteins, proteolytic protein inhibitors,semaphorin a semaphorin receptors, serotonin transport proteins,serotonin uptake inhibitors, serotonin receptors, serpins, serpinreceptors, and tumor suppressors.

In certain applications, the modified high-transduction efficiencyvectors may comprise a nucleic acid segment that encodes a polypeptideselected from the group consisting of BDNF, CNTF, CSF, EGF, FGF, G-SCF,GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, TGF-B2,TNF, VEGF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18. Such therapeutic agentsmay be of human, murine, avian, porcine, bovine, ovine, feline, canine,equine, epine, caprine, lupine or primate origin.

Recombinant AAV vectors useful according to the invention includesingle-stranded (ss) or self-complementary (sc) AAV vectors.

The rAAV vectors of the present invention may also be within an isolatedmammalian host cell, including for example, human, primate, murine,feline, canine, porcine, ovine, bovine, equine, epine, caprine andlupine host cells. The rAAV vectors may be within an isolated mammalianhost cell such as a human endothelial, epithelial, vascular, liver,lung, heart, pancreas, intestinal, kidney, cardiac, cancer or tumor,muscle, bone, neural, blood, or brain cell.

Therapeutic Uses

Another aspect of the invention pertains to uses of the rAAV vectors andvirions of the invention for efficient transduction of cells, tissues,and/or organs of interest, and/or for use in gene therapy.

In one embodiment, the present invention provides a method fortransduction of cells, tissues, and/or organs of interest, comprisingintroducing into a cell, a composition comprising an effective amount ofa rAAV vector and/or virion of present invention.

In certain embodiments, rAAV vectors and virions of the invention areused for transduction of mammalian host cells, including for example,human, primate, murine, feline, canine, porcine, ovine, bovine, equine,epine, caprine and lupine host cells. In certain embodiments, the rAAVvectors and virions of the invention are used for transduction ofendothelial, epithelial, vascular, liver, lung, heart, pancreas,intestinal, kidney, muscle, bone, dendritic, cardiac, neural, blood,brain, fibroblast or cancer cells.

In one embodiment, cells, tissues, and/or organs of a subject aretransduced using the rAAV vectors and/or virions of the presentinvention.

The term “subject,” as used herein, describes an organism, includingmammals such as primates, to which treatment with the compositionsaccording to the present invention can be provided. Mammalian speciesthat can benefit from the disclosed methods of treatment include, butare not limited to, apes; chimpanzees; orangutans; humans; monkeys;domesticated animals such as dogs and cats; livestock such as horses,cattle, pigs, sheep, goats, and chickens; and other animals such asmice, rats, guinea pigs, and hamsters.

In addition, the present invention provides a method for treatment of adisease, wherein the method comprises administering, to a subject inneed of such treatment, an effective amount of a composition comprisingthe rAAV vector and/or virion of the invention.

The term “treatment” or any grammatical variation thereof (e.g., treat,treating, and treatment etc.), as used herein, includes but is notlimited to, alleviating a symptom of a disease or condition; and/orreducing, suppressing, inhibiting, lessening, ameliorating or affectingthe progression, severity, and/or scope of a disease or condition.

The term “effective amount,” as used herein, refers to an amount that iscapable of treating or ameliorating a disease or condition or otherwisecapable of producing an intended therapeutic effect.

The invention also provides for the use of a composition disclosedherein in the manufacture of a medicament for treating, preventing orameliorating the symptoms of a disease, disorder, dysfunction, injury ortrauma, including, but not limited to, the treatment, prevention, and/orprophylaxis of a disease, disorder or dysfunction, and/or theamelioration of one or more symptoms of such a disease, disorder ordysfunction. Exemplary conditions for which rAAV viral based genetherapy may find particular utility include, but are not limited to,cancer, diabetes, autoimmune disease, kidney disease, cardiovasculardisease, pancreatic disease, intestinal disease, liver disease,neurological disease, neuromuscular disorder, neuromotor deficit,neuroskeletal impairment, neurological disability, neurosensorydysfunction, stroke, .alpha.sub.1-antitrypsin (AAT) deficiency, Batten'sdisease, ischemia, an eating disorder, Alzheimer's disease, Huntington'sdisease, Parkinson's disease, skeletal disease and pulmonary disease.

The invention also provides a method for treating or ameliorating thesymptoms of such a disease, injury, disorder, or dysfunction in amammal. Such methods generally involve at least the step ofadministering to a mammal in need thereof, one or more of the rAAVvectors and virions of the present invention, in an amount and for atime sufficient to treat or ameliorate the symptoms of such a disease,injury, disorder, or dysfunction in the mammal.

Such treatment regimens are particularly contemplated in human therapy,via administration of one or more compositions either intramuscularly,intravenously, subcutaneously, intrathecally, intraperitoneally, or bydirect injection into an organ or a tissue of the subject under care.

The invention also provides a method for providing to a mammal in needthereof, a therapeutically-effective amount of the rAAV compositions ofthe present invention, in an amount, and for a time effective to providethe patient with a therapeutically-effective amount of the desiredtherapeutic agent(s) encoded by one or more nucleic acid segmentscomprised within the rAAV vector. Preferably, the therapeutic agent isselected from the group consisting of a polypeptide, a peptide, anantibody, an antigen binding fragment, a ribozyme, a peptide nucleicacid, a siRNA, an RNAi, an antisense oligonucleotide and an antisensepolynucleotide.

Pharmaceutical Compositions

The present invention also provides therapeutic or pharmaceuticalcompositions comprising the active ingredient in a form that can becombined with a therapeutically or pharmaceutically acceptable carrier.The genetic constructs of the present invention may be prepared in avariety of compositions, and may also be formulated in appropriatepharmaceutical vehicles for administration to human or animal subjects.

The rAAV molecules of the present invention and compositions comprisingthem provide new and useful therapeutics for the treatment, control, andamelioration of symptoms of a variety of disorders, and in particular,articular diseases, disorders, and dysfunctions, including for exampleosteoarthritis, rheumatoid arthritis, and related disorders.

The invention also provides compositions comprising one or more of thedisclosed rAAV vectors, expression systems, virions, viral particles; ormammalian cells. As described hereinbelow, such compositions may furthercomprise a pharmaceutical excipient, buffer, or diluent, and may beformulated for administration to an animal, and particularly a humanbeing. Such compositions may further optionally comprise a liposome, alipid, a lipid complex, a microsphere, a microparticle, a nanosphere, ora nanoparticle, or may be otherwise formulated for administration to thecells, tissues, organs, or body of a subject in need thereof. Suchcompositions may be formulated for use in a variety of therapies, suchas for example, in the amelioration, prevention, and/or treatment ofconditions such as peptide deficiency, polypeptide deficiency, peptideoverexpression, polypeptide overexpression, including for example,conditions which result in diseases or disorders such as cancers,tumors, or other malignant growths, neurological deficit dysfunction,autoimmune diseases, articular diseases, cardiac or pulmonary diseases,ischemia, stroke, cerebrovascular accidents, transient ischemic attacks(TIA); diabetes and/or other diseases of the pancreas; cardiocirculatorydisease or dysfunction (including, e.g., hypotension, hypertension,atherosclerosis, hypercholesterolemia, vascular damage or disease;neural diseases (including, e.g., Alzheimer's, Huntington's, Tay-Sach'sand Parkinson's disease, memory loss, trauma, motor impairment,neuropathy, and related disorders); biliary, renal or hepatic disease ordysfunction; musculoskeletal or neuromuscular diseases (including, e.g.,arthritis, palsy, cystic fibrosis (CF), amyotrophic lateral sclerosis(ALS), multiple sclerosis (MS), muscular dystrophy (MD), and such like).

In one embodiment, the number of rAAV vector and/or virion particlesadministered to a mammal may be on the order ranging from 10³ to 10¹³particles/ml, or any values therebetween, such as for example, about10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or 10¹³ particles/ml. In oneembodiment, rAAV vector and/or virion particles of higher than 10¹³particles/ml are be administered. The rAAV vectors and/or virions can beadministered as a single dose, or divided into two or moreadministrations as may be required to achieve therapy of the particulardisease or disorder being treated. In most rAAV-based gene therapyregimens, the inventors believe that a lower titer of infectiousparticles will be required when using the modified-capsid rAAV vectors,than compared to conventional gene therapy protocols.

In certain embodiments, the present invention concerns formulation ofone or more rAAV-based compositions disclosed herein in pharmaceuticallyacceptable solutions for administration to a cell or an animal, eitheralone or in combination with one or more other modalities of therapy,and in particular, for therapy of human cells, tissues, and diseasesaffecting man.

If desired, nucleic acid segments, RNA, DNA or PNA compositions thatexpress one or more of therapeutic gene products may be administered incombination with other agents as well, such as, e.g., proteins orpolypeptides or various pharmaceutically-active agents, including one ormore systemic or topical administrations of therapeutic polypeptides,biologically active fragments, or variants thereof. In fact, there isvirtually no limit to other components that may also be included, giventhat the additional agents do not cause a significant adverse effectupon contact with the target cells or host tissues. The rAAV-basedgenetic compositions may thus be delivered along with various otheragents as required in the particular instance. Such compositions may bepurified from host cells or other biological sources, or alternativelymay be chemically synthesized as described herein. Likewise, suchcompositions may further comprise substituted or derivatized RNA, DNA,siRNA, mRNA, tRNA, ribozyme, catalytic RNA molecules, or PNAcompositions and such like.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal,intra-articular, intramuscular administration and formulation.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically-useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the AAVvector-based therapeutic constructs in suitably formulatedpharmaceutical compositions disclosed herein either subcutaneously,intraocularly, intravitreally, parenterally, subcutaneously,intravenously, intracerebro-ventricularly, intramuscularly,intrathecally, orally, intraperitoneally, by oral or nasal inhalation,or by direct injection to one or more cells, tissues, or organs bydirect injection. The methods of administration may also include thosemodalities as described in U.S. Pat. No. 5,543,158; U.S. Pat. No.5,641,515 and U.S. Pat. No. 5,399,363 (each specifically incorporatedherein by reference in its entirety). Solutions of the active compoundsas freebase or pharmacologically acceptable salts may be prepared insterile water and may also suitably mixed with one or more surfactants,such as hydroxypropylcellulose. Dispersions may also be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations containa preservative to prevent the growth of microorganisms.

The pharmaceutical forms of the AAV-based viral compositions suitablefor injectable use include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions (U.S. Pat. No. 5,466,468, specificallyincorporated herein by reference in its entirety). In all cases the formmust be sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(e.g., glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and/or vegetable oils. Properfluidity may be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum oil such as mineral oil, vegetable oil such as peanut oil,soybean oil, and sesame oil, animal oil, or oil of synthetic origin.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers.

The compositions of the present invention can be administered to thesubject being treated by standard routes including, but not limited to,pulmonary, intranasal, oral, inhalation, parenteral such as intravenous,topical, transdermal, intradermal, transmucosal, intraperitoneal,intramuscular, intracapsular, intraorbital, intracardiac, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal injection. In preferredembodiments, the composition is administered via intranasal, pulmonary,or oral route.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activeAAV vector-delivered therapeutic polypeptide-encoding DNA fragments inthe required amount in the appropriate solvent with several of the otheringredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

The AAV vector compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

The amount of AAV compositions and time of administration of suchcompositions will be within the purview of the skilled artisan havingbenefit of the present teachings. It is likely, however, that theadministration of therapeutically-effective amounts of the disclosedcompositions may be achieved by a single administration, such as forexample, a single injection of sufficient numbers of infectiousparticles to provide therapeutic benefit to the patient undergoing suchtreatment. Alternatively, in some circumstances, it may be desirable toprovide multiple, or successive administrations of the AAV vectorcompositions, either over a relatively short, or a relatively prolongedperiod of time, as may be determined by the medical practitioneroverseeing the administration of such compositions.

Expression Vectors

The present invention contemplates a variety of AAV-based expressionsystems, and vectors. In one embodiment the preferred AAV expressionvectors comprise at least a first nucleic acid segment that encodes atherapeutic peptide, protein, or polypeptide. In another embodiment, thepreferred AAV expression vectors disclosed herein comprise at least afirst nucleic acid segment that encodes an antisense molecule. Inanother embodiment, a promoter is operatively linked to a sequenceregion that encodes a functional mRNA, a tRNA, a ribozyme or anantisense RNA.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depends directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the functional RNA to which itis operatively linked.

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini or blunt ends. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted and to the vector DNA. The vector and DNA segment are thenjoined by hydrogen bonding between the complementary homopolymeric tailsto form recombinant DNA molecules.

To express a therapeutic agent in accordance with the present inventionone may prepare a tyrosine-modified rAAV expression vector thatcomprises a therapeutic agent-encoding nucleic acid segment under thecontrol of one or more promoters. To bring a sequence “under the controlof” a promoter, one positions the 5′ end of the transcription initiationsite of the transcriptional reading frame generally between about 1 andabout 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter.The “upstream” promoter stimulates transcription of the DNA and promotesexpression of the encoded polypeptide. This is the meaning of“recombinant expression” in this context. Particularly preferredrecombinant vector constructs are those that comprise a rAAV vector.Such vectors are described in detail herein.

When the use of such vectors is contemplated for introduction of one ormore exogenous proteins, polypeptides, peptides, ribozymes, and/orantisense oligonucleotides, to a particular cell transfected with thevector, one may employ the rAAV vectors or the tyrosine-modified rAAVvectors disclosed herein by genetically modifying the vectors to furthercomprise at least a first exogenous polynucleotide operably positioneddownstream and under the control of at least a first heterologouspromoter that expresses the polynucleotide in a cell comprising thevector to produce the encoded peptide, protein, polypeptide, ribozyme,siRNA, RNAi or antisense oligonucleotide. Such constructs may employheterologous promoters that are constitutive, inducible, or evencell-specific promoters. Exemplary such promoters include, but are notlimited to, viral, mammalian, and avian promoters, including for examplea CMV promoter, a .beta.-actin promoter, a hybrid CMV promoter, a hybrid.beta.-actin promoter, an EF1 promoter, a U1a promoter, a U1b promoter,a Tet-inducible promoter, a VP16-LexA promoter, and such like.

The vectors or expression systems may also further comprise one or moreenhancers, regulatory elements, transcriptional elements, to alter oreffect transcription of the heterologous gene cloned in the rAAVvectors. For example, the rAAV vectors of the present invention mayfurther comprise at least a first CMV enhancer, a synthetic enhancer, ora cell- or tissue-specific enhancer. The exogenous polynucleotide mayalso further comprise one or more intron sequences.

Therapeutic Kits

The invention also encompasses one or more of the genetically-modifiedrAAV vector compositions described herein together with one or morepharmaceutically-acceptable excipients, carriers, diluents, adjuvants,and/or other components, as may be employed in the formulation ofparticular rAAV-polynucleotide delivery formulations, and in thepreparation of therapeutic agents for administration to a subject, andin particularly, to a human. In particular, such kits may comprise oneor more of the disclosed rAAV compositions in combination withinstructions for using the viral vector in the treatment of suchdisorders in a subject, and may typically further include containersprepared for convenient commercial packaging. As such, preferred animalsfor administration of the pharmaceutical compositions disclosed hereininclude mammals, and particularly humans. Other preferred animalsinclude murines, bovines, equines, porcines, canines, and felines. Thecomposition may include partially or significantly purified rAAVcompositions, either alone, or in combination with one or moreadditional active ingredients, which may be obtained from natural orrecombinant sources, or which may be obtainable naturally or eitherchemically synthesized, or alternatively produced in vitro fromrecombinant host cells expressing DNA segments encoding such additionalactive ingredients.

Therapeutic kits may also be prepared that comprise at least one of thecompositions disclosed herein and instructions for using the compositionas a therapeutic agent. The container means for such kits may typicallycomprise at least one vial, test tube, flask, bottle, syringe or othercontainer means, into which the disclosed rAAV composition(s) may beplaced, and preferably suitably aliquoted. Where a second therapeuticpolypeptide composition is also provided, the kit may also contain asecond distinct container means into which this second composition maybe placed. Alternatively, the plurality of therapeutic biologicallyactive compositions may be prepared in a single pharmaceuticalcomposition, and may be packaged in a single container means, such as avial, flask, syringe, bottle, or other suitable single container means.The kits of the present invention will also typically include a meansfor containing the vial(s) in close confinement for commercial sale,such as, e.g., injection or blow-molded plastic containers into whichthe desired vial(s) are retained.

AAV Capsid Proteins

Supramolecular assembly of 60 individual capsid protein subunits into anon-enveloped, T-1 icosahedral lattice capable of protecting a 4.7-kbsingle-stranded DNA genome is a critical step in the life-cycle of thehelper-dependent human parvovirus, adeno-associated virus2 (AAV2). Themature 20-nm diameter AAV2 particle is composed of three structuralproteins designated VP1, VP2, and VP3 (molecular masses of 87, 73, and62 kDa respectively) in a ratio of 1:1:18. Based on its symmetry andthese molecular weight estimates, of the 60 capsid proteins comprisingthe particle, three are VP1 proteins, three are VP2 proteins, andfifty-four are VP3 proteins.

Biological Functional Equivalents

Modification and changes to the structure of the polynucleotides andpolypeptides of wild-type rAAV vectors to provide the improved rAAVvirions as described in the present invention to obtain functional viralvectors that possess desirable characteristics, particularly withrespect to improved delivery of therapeutic gene constructs to selectedmammalian cell, tissues, and organs for the treatment, prevention, andprophylaxis of various diseases and disorders, as well as means for theamelioration of symptoms of such diseases, and to facilitate theexpression of exogenous therapeutic and/or prophylactic polypeptides ofinterest via rAAV vector-mediated gene therapy. As mentioned above, oneof the key aspects of the present invention is the creation of one ormore mutations into specific polynucleotide sequences that encode one ormore of the therapeutic agents encoded by the disclosed rAAV constructs.In certain circumstances, the resulting polypeptide sequence is alteredby these mutations, or in other cases, the sequence of the polypeptideis unchanged by one or more mutations in the encoding polynucleotide toproduce modified vectors with improved properties for effecting genetherapy in mammalian systems.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the polynucleotide sequences disclosed herein,without appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics(Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

DEFINITIONS

In accordance with the present invention, polynucleotides, nucleic acidsegments, nucleic acid sequences, and the like, include, but are notlimited to, DNAs (including and not limited to genomic or extragenomicDNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but notlimited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleicacid segments either obtained from natural sources, chemicallysynthesized, modified, or otherwise prepared or synthesized in whole orin part by the hand of man.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andcompositions similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and compositions are described herein. For purposes of thepresent invention, the following terms are defined below:

The term “promoter,” as used herein refers to a region or regions of anucleic acid sequence that regulates transcription.

The term “regulatory element,” as used herein, refers to a region orregions of a nucleic acid sequence that regulates transcription.Exemplary regulatory elements include, but are not limited to,enhancers, post-transcriptional elements, transcriptional controlsequences, and such like.

The term “vector,” as used herein, refers to a nucleic acid molecule(typically comprised of DNA) capable of replication in a host celland/or to which another nucleic acid segment can be operatively linkedso as to bring about replication of the attached segment. A plasmid,cosmid, or a virus is an exemplary vector.

The term “substantially corresponds to,” “substantially homologous,” or“substantial identity,” as used herein, denote a characteristic of anucleic acid or an amino acid sequence, wherein a selected nucleic acidor amino acid sequence has at least about 70 or about 75 percentsequence identity as compared to a selected reference nucleic acid oramino acid sequence. More typically, the selected sequence and thereference sequence will have at least about 76, 77, 78, 79, 80, 81, 82,83, 84 or even 85 percent sequence identity, and more preferably atleast about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequenceidentity. More preferably still, highly homologous sequences often sharegreater than at least about 96, 97, 98, or 99 percent sequence identitybetween the selected sequence and the reference sequence to which it wascompared.

The percentage of sequence identity may be calculated over the entirelength of the sequences to be compared, or may be calculated byexcluding small deletions or additions which total less than about 25percent or so of the chosen reference sequence. The reference sequencemay be a subset of a larger sequence, such as a portion of a gene orflanking sequence, or a repetitive portion of a chromosome. However, inthe case of sequence homology of two or more polynucleotide sequences,the reference sequence will typically comprise at least about 18-25nucleotides, more typically at least about 26 to 35 nucleotides, andeven more typically at least about 40, 50, 60, 70, 80, 90, or even 100or so nucleotides.

Desirably, which highly homologous fragments are desired, the extent ofpercent identity between the two sequences will be at least about 80%,preferably at least about 85%, and more preferably about 90% or 95% orhigher, as readily determined by one or more of the sequence comparisonalgorithms well-known to those of skill in the art, such as e.g., theFASTA program analysis described by Pearson and Lipman (Proc. Natl.Acad. Sci. USA, 85(8):2444-8, April 1988).

The term “operably linked,” as used herein, refers to that the nucleicacid sequences being linked are typically contiguous, or substantiallycontiguous, and, where necessary to join two protein coding regions,contiguous and in reading frame. However, since enhancers generallyfunction when separated from the promoter by several kilobases andintronic sequences may be of variable lengths, some polynucleotideelements may be operably linked but not contiguous.

Materials and Methods

Cells and Antibodies.

HEK293, HeLa, NIH3T3 cells were obtained from the American Type CultureCollection and maintained as monolayer cultures in DMEM (Invitrogen)supplemented with 10% FBS (Sigma) and antibiotics (Lonza).Leukapheresis-derived peripheral blood mononuclear cells (PBMCs)(AllCells) were purified on Ficoll-Paque (GEHeathCare), resuspended inserum-free AIM-V medium (Lonza), and semi-adherent cell fractions wereincubated for 7 days with recombinant human IL-4 (500 U/mL) and GM-CSF(800 U/mL) (R&D Systems). Cell maturation was initiated with cytokinemixture including 10 ng/mL TNF-α, 10 ng/mL IL-1, 10 ng/mL IL-6, and 1mg/mL PGE2 (R&D Systems) for 48 hrs. Prior to EGFP expression cells werecharacterized for co-stimulatory molecules expression to ensure thatthey met the typical phenotype of mature dendritic cells (mDC) (CD80,RPE, murine IgG1; CD83, RPE, murine IgG1; CD86, FITC, murine IgG1;Invitrogen).

Site-Directed Mutagenesis.

A two-stage PCR was performed with plasmid pACG2 as described previously[Wang et al. (1999)] using Turbo Pfu Polymerase (Stratagen). Briefly, instage one, two PCR extension reactions were performed in separate tubesfor the forward and reverse PCR primer for 3 cycles. In stage two, thetwo reactions were mixed and a PCR reaction was performed for anadditional 15 cycles, followed by Dpn I digestion for 1 hr. Primers weredesigned to introduce changes from serine (TCA or AGC) to valine (GTA orGTC) for each of the residues mutated.

Production of Recombinant AAV Vectors.

Recombinant AAV2 vectors containing the EGFP gene driven by the chicken{circle around (r)}-actin promoter were generated as describedpreviously [Zolotukhin et al. (2002)]. Briefly, HEK293 cells weretransfected using Polyethelenimine (PEI, linear, MW 25,000, Polyscinces,Inc.). Seventy-two hrs post transfection, cells were harvested andvectors were purified by iodixanol (Sigma) gradient centrifugation andion exchange column chromatography (HiTrap Sp Hp 5 ml, GE Healthcare).Virus was then concentrated and the buffer was exchanged in three cyclesto lactated Ringer's using centrifugal spin concentrators (Apollo,150-kDa cut-off, 20-ml capacity, CLP) [Cheng et al. (2011)]. Ten μl ofpurified virus was treated with DNAse I (Invitrogen) for 2 hrs at 37°C., then additional 2 hrs with proteinase K (Invitrogen) at 56° C. Thereaction mixture was purified by phenol/chloroform, followed bychloroform treatment. Packaged DNA was precipitated with ethanol in thepresence of 20 μg glycogen (Invitrogen). DNAse I-resistant AAV particletiters were determined by RT-PCR with the following primers-pair,specific for the CBA promoter: forward 5′-TCCCATAGTAACGCCAATAGG-3′ (SEQID NO:11), reverse 5′-CTTGGCATATGATACACTTGATG-3′ (SEQ ID NO:12) and SYBRGreen PCR Master Mix (Invitrogen) [Aslanidi et al.].

Recombinant AAV Vector Transduction Assays In Vitro.

HEK293 or monocyte-direved dendritic cells (moDCs), were transduced withAAV2 vectors with 1,000 vgs/cell or 2,000 vgs/cell respectively, andincubated for 48 hrs. Alternatively, cells were pretreated with 50 μM ofselective serine/threonine kinases inhibitors2-(2-hydroxyethylamino)-6-aminohexylcarbamic acid tert-butylester-9-isopropylpurine (for CaMK-II), anthra[1,9-cd]pyrazol-6(2H)-one,1,9-pyrazoloanthrone (for JNK), and4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole(for MAPK) (CK59, JNK inhibitor 2, PD 98059, Calbiochem), 1 hr beforetransduction. Transgene expression was assessed as the total area ofgreen fluorescence (pixel²) per visual field (mean±SD) or by flowcytometry as described previously [Markusic et al. (2011) andJayandharan et al. (2011)]. Analysis of variance was used to comparetest results and the control, which were determined to be statisticallysignificant.

Western Blot Analysis.

Western blot analysis was performed as described previously [Aslanidi etal. (2007)]. Cells were harvested by centrifugation, washed with PBS,and resuspended in lysis buffer containing 50 mM Tris_HCl, pH 7.5, 120mM NaCl, 1% Nonidet P-40, 10% glycerol, 10 mM Na₄P₂O₇, 1 mMphenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA, and 1 mM EGTAsupplemented with protease and phosphotase inhibitors mixture (Set 2 and3, Calbiochem). The suspension was incubated on ice for 1 hr andclarified by centrifugation for 30 min at 14,000 rpm at 4° C. Followingnormalization for protein concentration, samples were separated using12% polyacrylamide/SDS electrophoresis, transferred to a nitrocellulosemembrane, and probed with primary antibodies, anti p-p38 MAPK(Thr180/Tyr182) rabbit mAb, total p38 MAPK rabbit mAb and GAPDH rabbitmAb (1:1000, CellSignaling), follow by secondary horseradishperoxidase-linked linked antibodies (1:1000, CellSignaling).

Specific Cytotoxic T-Lymphocytes Generation and Cytotoxicity Assay.

Monocytes-derived dendritic cells (moDCs) were generated as describedabove. Immature DCs were infected with AAV2-S662V vectors encoding humantelomerase cDNA (a generous gift from Dr. Karina Krotova, University ofFlorida), separated into two overlapping ORF-hTERT₈₃₈₋₂₂₂₉ andhTERT₂₀₄₂₋₃₄₅₄ at MOI 2,000 vgs/cell of each. Cells were then allowed toundergo stimulation with supplements to induce maturation. After 48 h,the mature DCs expressing hTERT were harvested and mixed with the PBMCsat a ratio of 20:1. CTLs were cultured in AIM-V medium containingrecombinant human IL-15 (20 IU/ml) and IL-7 (20 ng/ml) at 20×10⁶ cellsin 25 cm² flasks. Fresh cytokines were added every 2 days. After 7 dayspost-priming, the cells were harvested and used for killing assays[Heiser et al. (2002)]. A killing curve was generated and specific celllysis was determined by FACS analysis of live/dead cell ratios asdescribed previously [Mattis et al. (1997)]. Human immortalizedmyelogenous leukemia cell line, K562, was used as a target.

Statistical Analysis.

Results are presented as mean±S.D. Differences between groups wereidentified using a grouped-unpaired two-tailed distribution of Student'sT-test. P-values <0.05 were considered statistically significant.

EXAMPLES

Following are examples that illustrate procedures and embodiments forpracticing the invention. The examples should not be construed aslimiting.

Example 1—Inhibition of Specific Cellular Serine/Threonine KinaseIncreases the Transduction Efficiency of Recombinant AAV2 Vectors

The present inventors have demonstrated that inhibition of cellularepidermal growth factor receptor protein tyrosine kinase (EGFR-PTK)activity, as well as site-directed mutagenesis of the 7 surface-exposedtyrosine residues significantly increases the transduction efficiency ofAAV2 vectors by preventing phosphorylation of these residues, therebycircumventing ubiquitination and subsequent proteasome-mediateddegradation of the vectors. AAV2 capsids also contain 15 surface-exposedserine residues, which can potentially be phosphorylated by cellularserine/threonine kinases widely expressed in various cell types andtissues.

To examine whether inhibition of such kinase activity can preventphosphorylation of surface-exposed serine residues, and thus, improveintracellular trafficking and nuclear transport of AAV2 vectors, severalcommercially available specific inhibitors of cellular serine/threoninekinases, such as calmodulin-dependent protein kinase II (CamK-II), c-JunN-terminal kinase (JNK), and mitogen-activated protein kinase (p38MAPK), were used. HEK293 cells were pre-treated with specificinhibitors, such as 2-(2-hydroxyethylamino)-6-aminohexylcarbamic acidtert-butyl ester-9-isopropylpurine (for CaMK-II),anthra[1,9-cd]pyrazol-6(2H)-one, 1,9-pyrazoloanthrone (for JNK), and4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole(for p38 MAPK) for 1 h at various concentrations. Cells weresubsequently transduced with either single-stranded (ss) orself-complementary (sc) AAV2 vectors at 1,000 vector genomes (vgs) percell.

These results show that all inhibitors at a concentration of 50 μMsignificantly increased the transduction efficiency of both ssAAV2 andscAAV2 vectors, the p38 MAPK inhibitor being the most effective (FIG.3A-B). The results indicate that the increase in the transductionefficiency was due to prevention of phosphorylation of vector capsidsrather than improved viral second-strand DNA synthesis.

Example 2—Site-Directed Mutagenesis of Surface-Exposed Serine Residueson AAV2 Capsid Improves AAV2 Vector-Mediated Transgene Expression

The AAV2 capsid contains 50 serine (S) residues in the viral protein 3(VP3) common region of the three capsid VPs, of which 15 residues (S261,S264, S267, S276, S384, S458, S468, S492, S498, S578, S658, S662, S668,S707, S721) are surface-exposed. Each of the 15 S residues wassubstituted with valine (V) residues by site-directed mutagenesis asdescribed previously. Most mutants could be generated at titers similarto the WT AAV2 vectors, with the exception of S261V, S276V, and S658V,which were produced at ˜10 times lower titers, and S267V and S668V,which produced no detectible levels of DNAse I-resistant vectorparticles. The titers of S468V and S384V mutants were ˜3-5 times higherthan the WT AAV2 vectors. Each of the S-V mutant vectors was evaluatedfor transduction efficiency in 293 cells.

These results, shown in FIG. 4, indicate that of the 15 mutants, theS662V mutant transduced 293 cells ˜20-fold more efficiently than its WTcounterpart. The transduction efficiency of the S458V and the S492Vmutant vectors was increased by ˜4- and 2-fold, respectively. Thetransduction efficiency of the S468V and the S384V mutants, which wereproduced at titers higher than the WT AAV2 vectors, either remainedunchanged (S468V), or reduced ˜10-fold (S384V) at the same multiplicityof infection (MOI). The transduction efficiency of various serine-valinemutate AAV2 vectors is summarized in FIG. 9. In addition, no furtherincrease in the transduction efficiency was observed with thedouble-mutants (S458+662V and S492+662V), or the triple-mutant(S458+492+662V).

Example 3—Substitution of the Serine Residue at Position 662 withDifferent Amino Acids

In addition to S-to-V substitution at position 662, the following 7mutants with different amino acids: S662→Alanine (A), S662→Asparagine(N), S662→Aspartic acid (D), S662→Histidine (H), S662→Isoleucine (I),S662→Leucine (L), and S662→Phenylalanine (F) were also generated.Transduction efficiency of the mutant vectors was evaluated in 293cells.

The results, as shown in FIG. 5 and summarized in FIG. 10, demonstratethat the substitution of S with V led to production of the mostefficient mutant without any change in vector titers, when compared toother mutants. Replacement of S with N, I, L, or F decreased thepackaging efficiency ˜10-fold with no significant effect on thetransduction efficiency, whereas substitution with D or H increased thetransduction efficiency ˜8-fold and ˜4-fold, respectively, with noeffect on vector titers. Substitution of S to A increased the viraltiter up to ˜5-fold, and enhanced the transgene expression ˜3-foldcompared with the WT AAV2 vector. The observed variability in titers andinfectivity of the serine-mutants at position 662 suggests the criticalrole each of the amino acids plays in modulating both the AAV2 packagingefficiency and its biological activity.

Example 4—Transduction Efficiency of the S662V Vectors Correlates withthe P38 MAPK Activity in Various Cell Types

The S662V vector-mediated transgene expression is examined using thefollowing cells types: (i) NIH3T3 (mouse embryonic fibroblasts), (ii)H2.35 (mouse fetal hepatocytes), (iii) HeLa (human cervical cancercells), and (iv) primary human monocyte-derived dendritic cells (moDCs).These cell types were transduced with WT scAAV2-EGFP or S662VscAAV2-EGFP vectors at an MOI of 2,000 vgs per cell under identicalconditions. EGFP gene expression was evaluated 48 hrs post-infection(p.i.) for HeLa, 293 and moDCs, and 5 days p.i. for H2.35 and NIH3T3cells.

The results, as shown in FIG. 6A, show that although the absolutedifferences in the transduction efficiency between WT and S662V mutantvectors ranged from ˜3-fold (in H2.35 cells) to ˜20-fold (in 293 cells),the mutant vector was consistently more efficient in each cell typetested.

To examine whether the observed differences in the transductionefficiency of the WT and the mutant vectors is due to variations in thelevels of expression and/or activity of the cellular p38 MAPK, celllysates prepared from each cell type were analyzed on Western blotsprobed with specific antibodies to detect both total p38 MAPK andphospho-p38 MAPK levels. GAPDH was used as a loading control.

The results, as shown in FIG. 6B, indicate that while the p38 MAPKprotein levels were similar, the kinase activity, as determined by thelevel of phosphorylation, varied significantly among different celltypes, and the transduction efficiency of the S662V mutant vectorcorrelated roughly with the p38 MAPK activity. The results show p38MAPK-mediated phosphorylation of AAV2 vectors. In addition, transductionby the WT-AAV2 vectors did not lead to up-regulation of phosphorylationof p38 MAPK in either 293 cells or in moDCs; this indicates that AAVdoes not induce robust phenotypic changes in moDCs.

Example 5—S662V Mutant Vector-Mediated Transduction of Primary HumanMonocyte-Derived Dendritic Cell (moDCs) does not Lead to PhenotypicAlterations

MAPK family members play important roles in the development andmaturation of APCs. In this Example, moDCs, isolated from healthy donorleukapheresis, were treated with 50 μM selective kinase inhibitors asdescribed above and then transduced with WT scAAV2-EGFP vectors. Two hrsp.i., cells were treated with supplements (TNF-α, IL-1β, Il-6, PGE2) toinduce maturation. EGFP transgene expression was evaluated 48 hrs p.i.by fluorescence microscopy.

The results show that pre-treatment of moDCs with specific inhibitors ofJNK and p38 MAPK increased EGFP expression levels ˜2-fold and ˜3-fold,respectively, and the transduction efficiency was enhanced by ˜5-foldwith the S662V mutant vectors (FIG. 7).

Since inhibition of these kinases has previously been reported toprevent maturation of dendritic cells, the capability of S662V mutant toinduce phenotypic changes in DCs was also examined. Briefly, moDC wereinfected with increasingly higher MOI of up to 50,000 vgs per cell,harvested at 48 hrs p.i., and analyzed by fluorescence-activated cellsorting (FACS) for up regulation of surface co-stimulatory molecules.Flow cytometric analyses of DC maturation markers such as CD80, CD83 andCD86 indicated that, similar to WT AAV2 vectors, the S662V mutantvectors also did not induce the maturation of moDCs (FIG. 7C). Theresults show that AAV vectors have low immunogenicity.

Example 6—Generation of Human Telomerase (hTERT) Specific CytotoxicT-Lymphocyte (CTL) by moDC Transduced with AAV2-S662V Vectors

As the serine-mutant AAV2 vector-mediated transgene expression in moDCwas significantly improved compared with the WT-AAV2 vectors, thisExample evaluates the ability of S662V-loaded moDCs to stimulate thegeneration of cytotoxic T-lymphocytes and effective specific killing oftarget cells. Given that human telomerase is recognized as a uniqueanti-cancer target commonly expressed in most cancer cells, a truncatedhuman telomerase (hTERT) gene under the control of the chicken β-actinpromoter was cloned and the DNA was packaged into the AAV2 S662V mutant.Non-adherent peripheral blood mononuclear cells (PBMC) containing up to25% of CD8 positive cells were stimulated once with moDC/hTERT deliveredby the S662V vector. An immortalized myelogenous leukemia cell line,K562, was used for a two-color fluorescence assay of cell-mediatedcytotoxicity to generate a killing curve with subsequently reducedeffector to target cell ratio.

The result, as shown in FIG. 8, indicate that moDC loaded with hTERT caneffectively stimulate specific T cell clone proliferation and killingactivity compared with moDC expressing GFP. The results indicate thatAAV-based delivery methods can be used for vaccination.

Example 7—High-Efficiency AAV2 Vectors Obtained by Site-DirectedMutagenesis of Surface-Exposed Tyrosine, Serine, and/or ThreonineResidues

Adeno-associated virus vectors are currently in use in a number ofclinical trials as a delivery vehicle to target a variety of tissues toachieve sustained expression of therapeutic genes. However, large vectordoses are needed to observe therapeutic benefits. Production ofsufficient amounts of the vector also poses a challenge, as well as therisk of initiating an immune response to the vector. Thus, it iscritical to develop novel AAV vectors with high transduction efficiencyat lower doses.

The cellular epidermal growth factor receptor protein tyrosine kinase(EGFR-PTK) negatively impacts transgene expression from recombinant AAV2vectors primarily due to phosphorylation of AAV2 capsids at tyrosineresidues. Tyrosine-phosphorylated capsids are subsequently degraded bythe host proteasome machinery, which negatively impacts the transductionefficiency of AAV vectors. Selective inhibitors of JNK and p38 MAPKserine/threonine kinases improve the transduction efficiency, indicatingthat phosphorylation of certain surface-exposed serine or/and threonineresidues decreases the transduction efficiency of AAV vectors.

Site-directed mutagenesis to the capsid protein of the wild-type AAV2was performed. As shown in FIGS. 11 and 12, the serine (S662V) andthreonine (T491V) mutants of the wild-type AAV2 capsid proteinsubstantially increase the transduction efficiency of AAV vectors.

The serine (S662V) and threonine (T491V) mutations were combined withthe best-performing single (Y730F) and triple (Y730F+500+444F)tyrosine-mutants to generate the following vectors: (i) three double(S662V+T491V; Y730F+S662V; Y730F+T491V); (ii) one triple(S662V+Y730F+T491V); (iii) two quadruple (Y730+500+444F+S662V;Y730+500+44F+T491V); and (iv) one quintuple(Y730+500+4440F+S662V+T491V). The transduction efficiency of each of themutant vector was evaluated using a primary murine hepatocyte cell lineH3.25.

As shown in FIG. 13, the quadruple mutant (Y730+500+730F+T491V) to thewild-type AAV2 vector increased the transduction efficiency toapproximately 30-fold over that of the wild-type (WT) AAV2 vectors, andapproximately 3-fold over the Y730+500+444F mutant vector. Combining theS662V mutation with either the single (Y730F)- or the triple-tyrosinemutant (Y730F+500+444F) vector, negatively affected the transductionefficiency.

Genetically modified dendritic cells (DCs) have been extensivelystudied, and numerous Phase I and II clinical trials evaluating theirefficacy in patients with cancer have been initiated. However, currentmethods for DC loading are inadequate in terms of cell viability,uncertainty regarding the longevity of antigen presentation, and therestriction by the patient's haplotype. Successful transduction ofdifferent subsets of DCs by different commonly used serotypes of AAVvectors has been demonstrated and the potential advantage of anAAV-based antitumor vaccine discussed. However, further improvements ingene transfer by recombinant AAV vectors to DCs in terms of specificityand transduction efficiency are warranted to achieve a significantimpact when used as an anti-tumor vaccine.

Serine/threonine protein kinases can negatively regulate the efficiencyof recombinant AAV vector-mediated transgene expression byphosphorylating the surface-exposed serine and/or threonine residues onthe viral capsid and target the vectors for proteasome-mediateddegradation. Prevention of phosphorylation of the surface-exposed serineand threonine residues could allow the vectors to evade phosphorylationand subsequent ubiquitination and, thus, prevent proteasomaldegradation.

Site-directed mutagenesis was performed to the wild-type AAV vector ofeach of the 15 surface-exposed serine (S) residues. The results showthat substitution of S662 to valine (V) increased the transductionefficiency of the S662V mutant up to 6-fold, when compared to thewild-type AAV2 vector. In addition, site-directed mutagenesis wasperformed to substitute each of the 17 surface-exposed threonine (T)residues of the wild-type AAV2 vector with V (T251V, T329V, T330V,T454V, T455V, T503V, T550V, T592V, T581V, T597V, T491V, T671V, T659V,T660V, T701V, T713V, T716V). The transduction efficiency of each of theT-V mutant vectors was evaluated using primary human monocyte-deriveddendritic cells (moDCs) at an MOI of 2,000 vgs/cell. Followingmaturation with a cytokine mixture including 10 ng/mL a, 10 ng/mL IL-1,10 ng/mL IL-6, and 1 mg/mL PGE2, EGFP expression was analyzed 48 hrspost-infection under a fluorescent microscope. Cells were characterizedfor expression of co-stimulatory molecules (CD80, CD83, and CD86) toensure that they met the typical phenotype of mature dendritic cells(mDCs).

As shown in FIG. 14, mutations of the following T residues (T455V,T491V, T550V, T659V, T671V) increased the transduction efficiency ofmoDCs up to 5-fold, and the T491V mutant has the highest transductionefficiency.

To examine whether multiple mutations of T residues could furtherenhance the transduction efficiency, the following AAV2 mustants weregenerated: (i) four AAV2 vectors with double mutations with respect tothe wild-type AAV2 vector (T455V+T491V; T550V+T491V; T659V+T491V;T671V+T491V); (ii) two triple AAV2 vectors with (T455V+T491V+T550V;T550V+T659V+T491V) mutations with respect to the wild-type AAV2 vector;and (iii) one AAV2 vector with quadruple (T455V+T550V+T659V+T491V)mutations with respect to the wild-type AAV2 vector. Severalmultiple-mutant vectors increased the transduction efficiency ofdendritic cells, and the triple-mutant (T550V+T659V+T491V) wasidentified to be optimal, which increased the transduction efficiencyapproximately ten-fold compared with the wild-type (WT) AAV2 vector.Combining the best performing S662V mutant with T491V further enhancedthe transduction efficiency by approximately 8-fold.

Example 8—Targeted Mutagenesis of Ubiquitin-Binding Lysine Residues onthe Adeno-Associated Virus (AAV) Serotype 2 Capsid Improves itsTransduction Efficiency

It is now well recognized that hepatic gene transfer of high doses ofAAV vectors predispose to a robust adaptive immune response, from thedata available from hemophilia clinical trials. Thus, there is a need todevelop novel strategies which will allow lower doses of vectors to beused to achieve sustained phenotypic correction and limit vector relatedimmune-toxicities.

This Example shows that surface-exposed lysine residues of the VP3region of the VVA2 capsid protein are direct targets for host ubiquitinligases, and mutagenesis of these lysine residues improves transductionefficiency of the AAV vectors.

In silico analysis using an ubiquitination prediction software (UbPred)identified seven lysine residues (K39, K137, K143, K161, K490, K527 andK532) of the wild-type AAV2 capsid could be ubiquitinated. Lysine toArginine mutations in AAV2 Rep/Cap coding plasmid was carried out andhighly purified stocks of a recombinant self-complementary AAV2 vectorsexpressing EGFP [scAAV-CBa-EGFP] were generated in each of the sevenlysine mutant plasmids. The physical particle titres of lysine mutantvectors was comparable to wild-type (WT) scAAV vectors (˜0.5-1×10^12vgs/mL), suggesting that these mutations do not affect the structure orpackaging ability of mutant capsids.

scAAV vectors containing WT or each of the seven lysine mutant capsidswere then evaluated for their transduction potential in vitro.Approximately 8×10⁴ HeLa or HEK293 cells were mock-infected or infectedwith AAV at different multiplicities of infection (MOI, 500, 2000 or5000 vgs/cell). Forty-eight hours post-infection, transgene (EGFP)expression was measured by fluorescence microscopy and byflow-cytometry.

The results (FIG. 15) show that the K532R mutant vector significantlyincreased gene expression in both HeLa (18×) and HEK 293 (9×) cells invitro, when compared to the WT-AAV2 vector. The increased transductionefficacy of the K532R vector was consistent across three different MOIstested, with an average increase of 10-fold over the WT vector.

Example 9—AAV Vector-Mediated Activation of Canonical and AlternativeNF-κB Pathways In Vivo: Implications for Innate and Adaptive ImmuneResponses and Gene Therapy

Infection of HeLa cells with adeno-associated viral (AAV) vectors invitro results in activation of the alternative pathway of NF-κB, acentral regulator of cellular immune and inflammatory responses. Inaddition, activation of the alternative, but not the canonical pathway,regulates AAV-mediated transgene expression in these cells.

This Example examines the role of NF-κB in liver-directed AAV-mediatedgene transfer in mice. In vivo, AAV-mediated gene transfer results inconsecutive activation of the canonial and the alternative NF-κBpathways. These pathways are thought to drive primarily inflammation(canonical) or adaptive responses (alternative pathway). AAV2 vectorswith the wild-type (WT) or the tyrosine triple-mutant (TM) capsidsactivated the canonical NF-κB pathway within 2 hrs, resulting inexpression of pro-inflammatory cytokines and chemokines (FIG. 16A). Thistransient process is Toll-like receptor 9 (TLR9)-dependent, and likelyreflects the initial sensing of the vector genome by antigen-presentingcells. Western blot analyses (FIG. 16B) of liver homogenates prepared 9hrs post-vector delivery, showed abundance of the nuclear p52 proteincomponent of the alternative NF-κB pathway, likely resulting from genetransfer to hepatocytes.

Administration of the NF-κB inhibitor Bay11 prior to gene transfereffectively blocked activation of both pathways. This preventedpro-inflammatory innate immune responses and also dampened anti-AAVcapsid antibody formation (FIG. 16C). Importantly, Bay11 did notinterfere with long-term transgene expression mediated by both the WTand the TM AAV2 vectors (FIG. 16D).

The results show that transient immuno-suppression with NF-κB inhibitorprior to vector administration eliminates inflammation (caused by innateresponses), and also limits adaptive responses.

Example 10—Development of Optimized AAV3 Serotype Vectors: Mechanism ofHigh-Efficiency Transduction of Human Liver Cancer Cells

Of the 10 commonly used AAV serotypes, AAV3 has been reported totransduce cells and tissues poorly. However, the present inventorsdiscovered that AAV3 vectors transduce established human hepatoblastoma(HB) and human hepatocellular carcinoma (HCC) cell lines as well asprimary human hepatocytes extremely efficiently. AAV3 utilizes humanHGFR as a cellular receptor/co-receptor for viral entry.

This Example shows that both extracellular as well as intracellularkinase domains of hHGFR are involved in AAV3 vector entry andAAV3-mediated transgene expression. The results show that (i) AAV3vector-mediated transduction is significantly increased in T47D cells, ahuman breast cancer cell line that expresses undetectable levels of theendogenous hHGFR, following stable transfection and over-expression ofhHGFR (FIG. 17A); (ii) the tyrosine kinase activity associated withhHGFR negatively affects the transduction efficiency of AAV3 vectors(FIG. 17B,C); (iii) the use of proteasome inhibitors significantlyimproves AAV3 vector-mediated transduction; (iv) site-directedmutagenesis of specific surface-exposed tyrosine residues on the AAV3capsid leads to improved transduction efficiency; and (v) a specificcombination of two tyrosine-mutations further improves the extent oftransgene expression (FIG. 17D). These AAV3 vectors can be useful forthe gene therapy of liver cancer in humans.

Example 11—Site-Directed Mutagenesis of Surface-Exposed Lysine ResiduesLeads to Improved Transduction by Recombinant AAV2 and AAV8 Vectors inMurine Hepatocytes In Vivo

The ubiquitin-proteasome pathway plays a critical role in theintracellular trafficking of recombinant AAV2 vectors, which negativelyimpacts the transduction efficiency of these vectors. The primary signalfor ubiquitination is phosphorylation of specific surface-exposedtyrosine (Y), serine (S), and threonine (T) residues on the AAV2capsids; the removal of some of these residues significantly increasesthe transduction efficiency of the wild-type (WT) AAV2 vectors.

This Example shows that site-directed mutagenesis of surface-exposedlysine residues can prevent ubiquitination of AAV2 capsids, which inturn, could prevent vector degradation by the cellular proteasomalmachinery.

AAV2 vectors with a single mutation in the surface-exposed lysine (K)residues (K258, K490, K527, K532, K544, 549, and K556) with glutamicacid (E) are provided. The transduction efficiency of K490E, K544E,K549E, and K556E scAAV2 vectors expressing the EGFP reporter geneincreases up to 5-fold, when compared with WT AAV2 vectors (FIG. 21).K556E mutant has the highest transduction efficiency, with atransduction rate of 2,000 vgs/cell in vitro in Hela cells. Similarresults are obtained when 1×10¹⁰ vgs of each vector is deliveredintravenously to C57BL/6 mice in vivo, and transgene expression inhepatocytes is evaluated 2-weeks post-injections. Intravenous deliveryof 1×10¹⁰ vgs/animal of WT and K-mutant ssAAV2 vectors expressing thefirefly luciferase (Flue) reporter gene and bioluminescence imaging twoweeks post injection further corroborate these results.

Further, two of the most efficient mutants are combined to generate adouble-mutant (K544+556E). The transduction efficiency of thedouble-mutant ssAAV2-Fluc vectors in murine hepatocytes in vivoincreases by ˜2-fold compared with each of the single mutants, and˜10-fold compared with WT ssAAV2 vectors.

AAV8 vectors have previously been shown to transduce murine hepatocytesexceedingly well. As some of the surface-exposed K residues are alsoconserved in this serotype, ssAAV8-Flue vectors with K530E-, K547E-, orK569E-mutant are also generated. The transduction efficiency of K547Eand K569E ssAAV8-Flue vectors in murine hepatocytes in vivo increases by˜3-fold and ˜2-fold, respectively, when compared with WT ssAAV8 vectors(FIGS. 24 and 25).

The results, as shown in FIGS. 18-25, show that targeting thesurface-exposed lysine residues can be used to create efficient AAVserotype vectors for their potential use in human gene therapy.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and was set forth in its entirety herein.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

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We claim:
 1. An AAV VP3 protein comprising: a non-threonine amino acidresidue at a position that corresponds to T251, T330, T454, T455, T503,T550, T592, T491, T671, T659, T660, T701, T713, or T716 of the wild-typeAAV2 capsid protein of SEQ ID NO:2 wherein the non-threonine amino acidresidue is selected from valine (V), aspartic acid (D), or histidine(H).
 2. The AAV VP3 protein according to claim 1, further comprising anon-lysine amino acid residue at a position that corresponds to K459,K490, K532, K544, or K556 of the wild-type AAV2 capsid protein of SEQ IDNO:2.
 3. The AAV VP3 protein according to claim 1, further comprising anon-lysine amino acid residue at a position that corresponds to K530,K547, or K569 of the wild-type AAV8 capsid protein of SEQ ID NO:8. 4.The AAV VP3 protein according to claim 2, comprising non-lysine aminoacid residues at positions correspond to K544 and K556 of the wild-typeAAV2 capsid protein of SEQ ID NO:2.
 5. The AAV VP3 protein according toclaim 2, wherein the non-lysine amino acid residue is selected fromglutamic acid (E), arginine (R), serine (S), or isoleucine (I).
 6. TheAAV VP3 protein according to claim 1, further comprising a glutamic acid(E) amino acid residue at a position that corresponds to K258, K321,K459, K490, K507, K527, K572, K532, K544, K549, K556, K649, K655, orK706 of the wild-type AAV2 capsid protein of SEQ ID NO:2.
 7. The AAV VP3protein according to claim 4, comprising glutamic acid (E) amino acidresidues at positions correspond to K544 and K556 of the wild-type AAV2capsid protein of SEQ ID NO:2.
 8. The AAV VP3 protein according to claim3, comprising a glutamic acid (E) amino acid residue at a position thatcorresponds to K530, K547, or K569 of the wild-type AAV8 capsid proteinof SEQ ID NO:8.
 9. The AAV VP3 protein according to claim 1, furthercomprising a non-serine amino acid residue at a position thatcorresponds to S662 of the wild-type AAV2 capsid protein of SEQ ID NO:2.10. The AAV VP3 protein according to claim 9, wherein the non-serineamino acid residue is selected from valine (V), aspartic acid (D), orhistidine (H).
 11. The AAV VP3 protein according to claim 10, comprisinga valine residue at a position that corresponds to S662 of the wild-typeAAV2 capsid protein of SEQ ID NO:2.
 12. The AAV VP3 protein according toclaim 1, further comprising: (a) a non-lysine amino acid residue at aposition that corresponds to K258, K321, K459, K490, K507, K527, K572,K532, K544, K549, K556, K649, K655, K665, or K706 of the wild-type AAV2capsid protein of SEQ ID NO:2; (b) a non-lysine amino acid residue at aposition that corresponds to K530, K547, or K569 of the wild-type AAV8capsid protein of SEQ ID NO:8; (c) a non-serine amino acid residue at aposition that corresponds to 5261, 5264, 5267, S276, 5384, 5458, 5468,5492, 5498, 5578, 5658, 5662, 5668, 5707, or 5721 of the wild-type AAV2capsid protein of SEQ ID NO:2; (d) a second non-threonine amino acidresidue at a position that corresponds to T251, T329, T330, T454, T455,T503, T550, T592, T581, T597, T491, T671, T659, T660, T701, T713, orT716 of the wild-type AAV2 capsid protein of SEQ ID NO:2 or (e) anon-tyrosine amino acid residue at a position that corresponds to Y252,Y272, Y444, Y500, Y700, Y704, or Y730 of the wild-type AAV2 capsidprotein of SEQ ID NO:2.
 13. The AAV VP3 protein according to claim 1,further comprising a phenylalanine amino acid residue at a position thatcorresponds to Y252, Y272, Y444, Y500, Y700, Y704, or Y730 of thewild-type AAV2 capsid protein of SEQ ID NO:2.
 14. The AAV VP3 proteinaccording to claim 1, further comprising a non-threonine amino acidresidue at a position that corresponds to T329, T581, or T597 of thewild-type AAV2 capsid protein of SEQ ID NO:2 wherein the non-threonineamino acid residue is selected from valine (V), aspartic acid (D), orhistidine (H).
 15. A composition comprising (a) an AAV particlecomprising the AAV VP3 protein in accordance with claim 1, and (b) apharmaceutically-acceptable buffer, diluent, or vehicle.
 16. Thecomposition according to claim 15, wherein the AAV particle comprises aVP3 protein further comprising a non-lysine amino acid residue at aposition that corresponds to K459, K490, K532, K544, or K556 of thewild-type AAV2 capsid protein of SEQ ID NO:2.
 17. The compositionaccording to claim 15, further comprising a non-lysine amino acidresidue at a position that corresponds to K530, K547, or K569 of thewild-type AAV8 capsid protein of SEQ ID NO:8.
 18. The compositionaccording to claim 15, further comprising a glutamic acid (E) amino acidresidue at a position that corresponds to K258, K321, K459, K490, K507,K527, K572, K532, K544, K549, K556, K649, K655, or K706 of the wild-typeAAV2 capsid protein of SEQ ID NO:2.
 19. The composition according toclaim 15, further comprising a non-serine amino acid residue at aposition that corresponds to S662 of the wild-type AAV2 capsid proteinof SEQ ID NO:2.
 20. The AAV VP3 protein according to claim 1, whereinthe AAV VP3 protein is a recombinant AAV3 VP3 protein.
 21. Thecomposition according to claim 15, wherein the AAV VP3 protein is arecombinant AAV3 VP3 protein.
 22. The composition of claim 15, whereinthe rAAV particle in the composition further comprises a polynucleotidethat encodes a therapeutic agent and that is operably linked to apromoter.
 23. A method of transducing cells, comprising introducing intoa host cell, a composition comprising an effective amount of an AAVparticle comprising the AAV VP3 protein of claim
 1. 24. The methodaccording to claim 23, wherein the VP3 protein further comprises anon-lysine amino acid residue at a position that corresponds to K459,K490, K532, K544, or K556 of the wild-type AAV2 VP3 protein of SEQ IDNO:2 or K530, K547, or K569 of the wild-type AAV8 capsid protein of SEQID NO:8.
 25. The method according to claim 24, wherein the non-lysineamino acid residue is glutamic acid (E), arginine (R), serine (S), orisoleucine (I).
 26. The method according to claim 23, wherein the hostcell is a mammalian cell.
 27. The method according to claim 23, whereinthe host cell is a endothelial, epithelial, vascular, liver, lung,heart, pancreas, intestinal, kidney, muscle, bone, dendritic, cardiac,neural, blood, brain, fibroblast, or cancer cell.
 28. The methodaccording to claim 23, wherein the cell is in a subject.